The role of in vitro methods as alternatives to animals in toxicity testing.

PubMed

Anadón, Arturo; Martínez, María Aranzazu; Castellano, Victor; Martínez-Larrañaga, María Rosa

2014-01-01

It is accepted that animal testing should be reduced, refined or replaced as far as it is practicably possible. There are also a wide variety of in vitro models, which are used as screening studies and mechanistic investigations. The ability of an in vitro assay to be reliable, biomedically, is essential in pharmaceutical development. Furthermore, it is necessary that cells used in in vitro testing mimic the phenotype of cells within the human target tissue. The focus of this review article is to identify the key points of in vitro assays. In doing so, the authors take into account the chemical agents that are assessed and the integrated in vitro testing strategies. There is a transfer of toxicological data from primary in vivo animal studies to in vitro assays. The key element for designing an integrated in vitro testing strategy is summarized as follows: exposure modeling of chemical agents for in vitro testing; data gathering, sharing and read-across for testing a class of chemical; a battery of tests to assemble a broad spectrum of data on different mechanisms of action to predict toxic effects; and applicability of the test and the integrated in vitro testing strategies and flexibility to adjust the integrated in vitro testing strategies to test substance. While these methods will be invaluable if effective, more studies must be done to ensure reliability and suitability of these tests for humans.

Species-specific predictive models of developmental toxicity using the ToxCast chemical library

EPA Science Inventory

EPA’s ToxCastTM project is profiling the in vitro bioactivity of chemicals to generate predictive models that correlate with observed in vivo toxicity. In vitro profiling methods are based on ToxCast data, consisting of over 600 high-throughput screening (HTS) and high-content sc...

[The in vitro dissolution of total composition of the tablet of rhizomes of Ligusticum chuanxiong components and in vitro-in vivo correlation by the method of area under the absorbance-wavelength curve].

PubMed

Lai, Hong-qiang; Hu, Yue; Li, Xiao-dong

2015-06-01

To discuss the availability of evaluation on the dissolution studies of the multicomponents in traditional Chinese medicine, the in vitro dissolution of total composition of the tablet of rhizomes of Ligusticum chuanxiong components and its correlation with the in vivo were studied by the method of area under the absorbance-wavelength curve (AUAWC). Taken the tablet of rhizomes of Ligusticum chuanxiong components which is composed of sodium ferulate and ligustrazine hydrochloride as subject model, the dissolution tests were carried out with basket method. The plasma concentrations of tablets in different rats were determined by AUAWC at different interval times. The in vivo absorption percentage was calculated by Wagner-Nelson equation to evaluate the in vitro and in vivo correlation. According to the results, the cumulative dissolution in vitro of total composition of tablets of rhizomes of Ligusticum chuanxiong components at 60 min was 90.65% in water by AUAWC. The in vivo pharmacokinetics is fitted with an one-compartment model. The linear equation based on the cumulative dissolution rate (fr) and absorption percentage (fa) at 5, 10, 20, 30 and 60 min was fa = 0.819 7 fr+0.183 and the correlation coefficient was 0.959 5, which showed a good correlation between the in vitro dissolution and the in vivo absorption percentage. The method of AUAWC can be used accurately, feasibly and conveniently to evaluate the in vitro and in vivo correlation of total composition of tablets of rhizomes of Ligusticum chuanxiong components, which will provide better guidance to study the in vitro and in vivo correlation of sustained release preparation etc under complex system of traditional Chinese medicine in the future.

Toward the establishment of standardized in vitro tests for lipid-based formulations, part 1: method parameterization and comparison of in vitro digestion profiles across a range of representative formulations.

PubMed

Williams, Hywel D; Sassene, Philip; Kleberg, Karen; Bakala-N'Goma, Jean-Claude; Calderone, Marilyn; Jannin, Vincent; Igonin, Annabel; Partheil, Anette; Marchaud, Delphine; Jule, Eduardo; Vertommen, Jan; Maio, Mario; Blundell, Ross; Benameur, Hassan; Carrière, Frédéric; Müllertz, Anette; Porter, Christopher J H; Pouton, Colin W

2012-09-01

The Lipid Formulation Classification System Consortium is an industry-academia collaboration, established to develop standardized in vitro methods for the assessment of lipid-based formulations (LBFs). In this first publication, baseline conditions for the conduct of digestion tests are suggested and a series of eight model LBFs are described to probe test performance across different formulation types. Digestion experiments were performed in vitro using a pH-stat apparatus and danazol employed as a model poorly water-soluble drug. LBF digestion (rate and extent) and drug solubilization patterns on digestion were examined. To evaluate cross-site reproducibility, experiments were conducted at two sites and highly consistent results were obtained. In a further refinement, bench-top centrifugation was explored as a higher throughput approach to separation of the products of digestion (and compared with ultracentrifugation), and conditions under which this method was acceptable were defined. Drug solubilization was highly dependent on LBF composition, but poorly correlated with simple performance indicators such as dispersion efficiency, confirming the utility of the digestion model as a means of formulation differentiation. Copyright © 2012 Wiley Periodicals, Inc.

Probe molecules (PrM) approach in adverse outcome pathway (AOP) based high throughput screening (HTS): in vivo discovery for developing in vitro target methods

EPA Science Inventory

Efficient and accurate adverse outcome pathway (AOP) based high-throughput screening (HTS) methods use a systems biology based approach to computationally model in vitro cellular and molecular data for rapid chemical prioritization; however, not all HTS assays are grounded by rel...

Comparison of trout hepatocytes and liver S9 fractions as in vitro models for predicting hepatic clearance in fish

EPA Science Inventory

Isolated hepatocytes and liver S9 fractions have been used to collect in vitro biotransformation data for fish as a means of improving modeled estimates of chemical bioaccumulation. To date, however, there have been few direct comparisons of these two methods. In the present st...

Comparison of trout hepatocytes and liver S9 fractions as in vitro models for predicting hepatic clearance in fish

EPA Science Inventory

Isolated hepatocytes and liver S9 fractions have been used to collect in vitro biotransformation data for fish as a means of improving modeled estimates of chemical bioaccumulation. To date, however, there have been few direct comparisons of these two methods. In the present stud...

Toxicokinetic Triage for Environmental Chemicals

EPA Science Inventory

Toxicokinetic (TK) models are essential for linking administered doses to blood and tissue concentrations. In vitro-to-in vivo extrapolation (IVIVE) methods have been developed to determine TK from limited in vitro measurements and chemical structure-based property predictions, p...

Bone marrow-on-a-chip replicates hematopoietic niche physiology in vitro.

PubMed

Torisawa, Yu-suke; Spina, Catherine S; Mammoto, Tadanori; Mammoto, Akiko; Weaver, James C; Tat, Tracy; Collins, James J; Ingber, Donald E

2014-06-01

Current in vitro hematopoiesis models fail to demonstrate the cellular diversity and complex functions of living bone marrow; hence, most translational studies relevant to the hematologic system are conducted in live animals. Here we describe a method for fabricating 'bone marrow-on-a-chip' that permits culture of living marrow with a functional hematopoietic niche in vitro by first engineering new bone in vivo, removing it whole and perfusing it with culture medium in a microfluidic device. The engineered bone marrow (eBM) retains hematopoietic stem and progenitor cells in normal in vivo-like proportions for at least 1 week in culture. eBM models organ-level marrow toxicity responses and protective effects of radiation countermeasure drugs, whereas conventional bone marrow culture methods do not. This biomimetic microdevice offers a new approach for analysis of drug responses and toxicities in bone marrow as well as for study of hematopoiesis and hematologic diseases in vitro.

Modeling Biotransformation Using In Vitro Data on Parent-Metabolite Pairs within the ToxCast Phase I Chemical Set

EPA Science Inventory

A major focus in toxicology research is the development of new in vitro methods to predict in vivo chemical toxicity. Within the EPA ToxCast program, a broad range of in vitro biochemical and cellular assays have been deployed to profile the biological activity of 320 Phase I che...

In Vitro Effects of Cooking Methods on Digestibility of Lipids and Formation of Cholesterol Oxidation Products in Pork

PubMed Central

Moon, Sung Sil

2014-01-01

This study investigated the effects of cooking methods on the digestibility of lipids and formation of cholesterol oxidation products (COPs) in pork, during in vitro human digestion. Pork patties were cooked using four different methods (oven cooking, pan frying, boiling, and microwaving), to an internal temperature of approximately 85℃. The digestibility of pork patties were then evaluated, using the in vitro human digestion model that simulated the composition (pH, minerals, surfaceactive components, and enzymes) of digestive juices in the human mouth, stomach, and small intestine. The total lipid digestibility was higher after microwave cooking, whereas pan-frying resulted in lower in vitro digestibility, compared to the other cooking methods. The microwaving method followed by in vitro digestion also showed significantly higher content of free fatty acids and thiobarbituric acid reactive substances (TBARS), compared to the other cooking methods; whereas, the pan frying and boiling methods showed the lowest. Cholesterol content was not significantly different among the cooked samples before, and after in vitro human digestion. The formation of COPs was significantly higher in the microwave-treated pork samples, compared to those cooked by the other methods, which was consistent with the trend for lipid peroxidation (TBARS). We propose that from the point of view of COPs formation and lipid oxidation, the pan-frying or boiling methods would be useful. PMID:26761168

An Accelerated Release Method of Risperidone Loaded PLGA Microspheres with Good IVIVC.

PubMed

Hu, Xiaoqin; Zhang, Jianwei; Tang, Xuemei; Li, Mingyuan; Ma, Siyu; Liu, Cheng; Gao, Yue; Zhang, Yue; Liu, Yan; Yu, Fanglin; Yang, Yang; Guo, Jia; Li, Zhiping; Mei, Xingguo

2018-01-01

A long release period lasting several days or several weeks is always needed and thereby it is tedious and time consuming to screen formulations of such microspheres with so long release period and evaluate their release profiles in vitro with conventional long-term or "real-time" release method. So, an accelerated release testing of such system is necessary for formulation design as well as quality control purpose. The purpose of this study is to obtain an accelerated release method of risperidone loaded poly(lactic-co-glycolic acid) (PLGA) microspheres with good in vitro/in vivo correlation (IVIVC). Two formulations of risperidone loaded PLGA microspheres used for evaluating IVIVC were prepared by O/W method. The accelerated release condition was optimized by investigating the effect of pH, osmotic pressure, temperature and ethanol concentration on the release of risperidone from microspheres and the in vitro accelerated release profiles of risperidone from PLGA microspheres were obtained under this optimized accelerated release condition. The plasma concentration of risperidone were also detected after subcutaneous injection of risperidone loaded microspheres to rats. The in vivo cumulative absorption profiles were then calculated using Wagner-Nelson model, Loo- Riegelman model and numerical convolution model, respectively. The correlation between in vitro accelerated release and in vivo cumulative absorption were finally evaluated with Least Square Method. It was shown that temperature and ethanol concentration significantly affected the release of risperidone from the microspheres while pH and osmotic pressure of release media slightly affected the release behavior of risperidone. The in vitro release of risperidone from microspheres were finally undergone in PBS (pH7.0, 300mosm) with 20% (V/V) ethanol at 45°C. The sustained and complete release of risperidone was observed in both formulations under the accelerated release condition although these two release profiles were dissimilar. The correlation coefficients (R2) of IVIVC were all above 0.95 and the slopes were all between 0.9564 and 1.1868 in spite of fitted model and microsphere formulation. An in vitro accelerated release method of risperidone microspheres with good IVIVC was established in this paper and this accelerated release method was supposed to have great potential in both in vivo performance prediction and quality control for risperidone loaded PLGA microspheres. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Deriving Signatures of In Vivo Toxicity Using Both Efficacy and Potency Information from In Vitro Assays: Evaluating Model Performance as a Function of Increasing Variability in Experimental Data

EPA Science Inventory

The US EPA ToxCast program aims to develop methods for mechanistically-based chemical prioritization using a suite of high throughput, in vitro assays that probe relevant biological pathways, and coupling them with statistical and machine learning methods that produce predictive ...

An in vitro digestion method adapted for carotenoids and carotenoid esters: moving forward towards standardization.

PubMed

Rodrigues, Daniele Bobrowski; Mariutti, Lilian Regina Barros; Mercadante, Adriana Zerlotti

2016-12-07

In vitro digestion methods are a useful approach to predict the bioaccessibility of food components and overcome some limitations or disadvantages associated with in vivo methodologies. Recently, the INFOGEST network published a static method of in vitro digestion with a proposal for assay standardization. The INFOGEST method is not specific for any food component; therefore, we aimed to adapt this method to assess the in vitro bioaccessibility of carotenoids and carotenoid esters in a model fruit (Byrsonima crassifolia). Two additional steps were coupled to the in vitro digestion procedure, centrifugation at 20 000g for the separation of the aqueous phase containing mixed micelles and exhaustive carotenoid extraction with an organic solvent. The effect of electrolytes, enzymes and bile acids on carotenoid micellarization and stability was also tested. The results were compared with those found with a simpler method that has already been used for carotenoid bioaccessibility analysis. These values were in the expected range for free carotenoids (5-29%), monoesters (9-26%) and diesters (4-28%). In general, the in vitro bioaccessibility of carotenoids assessed by the adapted INFOGEST method was significantly higher (p < 0.05) than those assessed by the simplest protocol, with or without the addition of simulated fluids. Although no trend was observed, differences in bioaccessibility values depended on the carotenoid form (free, monoester or diester), isomerization (Z/E) and the in vitro digestion protocol. To the best of our knowledge, it was the first time that a systematic identification of carotenoid esters by HPLC-DAD-MS/MS after in vitro digestion using the INFOGEST protocol was carried out.

Non-invasive Assessments of Adipose Tissue Metabolism In Vitro.

PubMed

Abbott, Rosalyn D; Borowsky, Francis E; Quinn, Kyle P; Bernstein, David L; Georgakoudi, Irene; Kaplan, David L

2016-03-01

Adipose tissue engineering is a diverse area of research where the developed tissues can be used to study normal adipose tissue functions, create disease models in vitro, and replace soft tissue defects in vivo. Increasing attention has been focused on the highly specialized metabolic pathways that regulate energy storage and release in adipose tissues which affect local and systemic outcomes. Non-invasive, dynamic measurement systems are useful to track these metabolic pathways in the same tissue model over time to evaluate long term cell growth, differentiation, and development within tissue engineering constructs. This approach reduces costs and time in comparison to more traditional destructive methods such as biochemical and immunochemistry assays and proteomics assessments. Towards this goal, this review will focus on important metabolic functions of adipose tissues and strategies to evaluate them with non-invasive in vitro methods. Current non-invasive methods, such as measuring key metabolic markers and endogenous contrast imaging will be explored.

Non-invasive assessments of adipose tissue metabolism in vitro

PubMed Central

Abbott, Rosalyn D.; Borowsky, Francis E.; Quinn, Kyle P.; Bernstein, David L.; Georgakoudi, Irene; Kaplan, David L.

2015-01-01

Adipose tissue engineering is a diverse area of research where the developed tissues can be used to study normal adipose tissue functions, create disease models in vitro, and replace soft tissue defects in vivo. Increasing attention has been focused on the highly specialized metabolic pathways that regulate energy storage and release in adipose tissues which affect local and systemic outcomes. Non-invasive, dynamic measurement systems are useful to track these metabolic pathways in the same tissue model over time to evaluate long term cell growth, differentiation, and development within tissue engineering constructs. This approach reduces costs and time in comparison to more traditional destructive methods such as biochemical and immunochemistry assays and proteomics assessments. Towards this goal, this review will focus on important metabolic functions of adipose tissues and strategies to evaluate them with noninvasive in vitro methods. Current non-invasive methods, such as measuring key metabolic markers and endogenous contrast imaging will be explored. PMID:26399988

Validation of an immortalized human (hBMEC) in vitro blood-brain barrier model.

PubMed

Eigenmann, Daniela Elisabeth; Jähne, Evelyn Andrea; Smieško, Martin; Hamburger, Matthias; Oufir, Mouhssin

2016-03-01

We recently established and optimized an immortalized human in vitro blood-brain barrier (BBB) model based on the hBMEC cell line. In the present work, we validated this mono-culture 24-well model with a representative series of drug substances which are known to cross or not to cross the BBB. For each individual compound, a quantitative UHPLC-MS/MS method in Ringer HEPES buffer was developed and validated according to current regulatory guidelines, with respect to selectivity, precision, and reliability. Various biological and analytical challenges were met during method validation, highlighting the importance of careful method development. The positive controls antipyrine, caffeine, diazepam, and propranolol showed mean endothelial permeability coefficients (P e) in the range of 17-70 × 10(-6) cm/s, indicating moderate to high BBB permeability when compared to the barrier integrity marker sodium fluorescein (mean P e 3-5 × 10(-6) cm/s). The negative controls atenolol, cimetidine, and vinblastine showed mean P e values < 10 × 10(-6) cm/s, suggesting low permeability. In silico calculations were in agreement with in vitro data. With the exception of quinidine (P-glycoprotein inhibitor and substrate), BBB permeability of all control compounds was correctly predicted by this new, easy, and fast to set up human in vitro BBB model. Addition of retinoic acid and puromycin did not increase transendothelial electrical resistance (TEER) values of the BBB model.

Heat effects on drug delivery across human skin

PubMed Central

Hao, Jinsong; Ghosh, Priyanka; Li, S. Kevin; Newman, Bryan; Kasting, Gerald B.; Raney, Sam G.

2016-01-01

Introduction Exposure to heat can impact the clinical efficacy and/or safety of transdermal and topical drug products. Understanding these heat effects and designing meaningful in vitro and in vivo methods to study them are of significant value to the development and evaluation of drug products dosed to the skin. Areas covered This review provides an overview of the underlying mechanisms and the observed effects of heat on the skin and on transdermal/topical drug delivery, thermoregulation and heat tolerability. The designs of several in vitro and in vivo heat effect studies and their results are reviewed. Expert opinion There is substantial evidence that elevated temperature can increase transdermal/topical drug delivery. However, in vitro and in vivo methods reported in the literature to study heat effects of transdermal/topical drug products have utilized inconsistent study conditions, and in vitro models require better characterization. Appropriate study designs and controls remain to be identified, and further research is warranted to evaluate in vitro-in vivo correlations and the ability of in vitro models to predict in vivo effects. The physicochemical and pharmacological properties of the drug(s) and the drug product, as well as dermal clearance and heat gradients may require careful consideration. PMID:26808472

[Improved device and method for determination of protein digestibility in vitro].

PubMed

Lipatov, N N; Iudina, S B; Lisitsyn, A B

1994-01-01

The ten-cells device for modelling of ferment hydrolysis of food proteins by acid basic proteases of human alimentary canal is described. The new procedure for the calculation of quantitative characteristic of proteins digestion "in vitro" is presented.

A comparative study on the stress distribution around dental implants in three arch form models for replacing six implants using finite element analysis.

PubMed

Zarei, Maryam; Jahangirnezhad, Mahmoud; Yousefimanesh, Hojatollah; Robati, Maryam; Robati, Hossein

2018-01-01

Dental implant is a method to replacement of missing teeth. It is important for replacing the missed anterior teeth. In vitro method is a safe method for evaluation of stress distribution. Finite element analysis as an in vitro method evaluated stress distribution around replacement of six maxillary anterior teeth implants in three models of maxillary arch. In this in vitro study, using ABAQUS software (Simulia Corporation, Vélizy-Villacoublay, France), implant simulation was performed for reconstruction of six maxillary anterior teeth in three models. Two implants were placed on both sides of the canine tooth region (A model); two implants on both sides of the canine tooth region and another on one side of the central incisor region (B model); and two implants on both sides of the canine tooth region and two implants in the central incisor area (C model). All implants evaluated in three arch forms (tapered, ovoid, and square). Data were analyzed by finite analysis software. Von Mises stress by increasing of implant number was reduced. In a comparison of A model in each maxillary arch, the stress created in the cortical and cancellous bones in the square arch was less than ovoid and tapered arches. The stress created in implants and cortical and cancellous bones in C model was less than A and B models. The C model (four-implant) reduced the stress distribution in cortical and cancellous bones, but this pattern must be evaluated according to arch form and cost benefit of patients.

Toxicity assessment of industrial chemicals and airborne contaminants: transition from in vivo to in vitro test methods: a review.

PubMed

Bakand, S; Winder, C; Khalil, C; Hayes, A

2005-12-01

Exposure to occupational and environmental contaminants is a major contributor to human health problems. Inhalation of gases, vapors, aerosols, and mixtures of these can cause a wide range of adverse health effects, ranging from simple irritation to systemic diseases. Despite significant achievements in the risk assessment of chemicals, the toxicological database, particularly for industrial chemicals, remains limited. Considering there are approximately 80,000 chemicals in commerce, and an extremely large number of chemical mixtures, in vivo testing of this large number is unachievable from both economical and practical perspectives. While in vitro methods are capable of rapidly providing toxicity information, regulatory agencies in general are still cautious about the replacement of whole-animal methods with new in vitro techniques. Although studying the toxic effects of inhaled chemicals is a complex subject, recent studies demonstrate that in vitro methods may have significant potential for assessing the toxicity of airborne contaminants. In this review, current toxicity test methods for risk evaluation of industrial chemicals and airborne contaminants are presented. To evaluate the potential applications of in vitro methods for studying respiratory toxicity, more recent models developed for toxicity testing of airborne contaminants are discussed.

MUSCLEMOTION: A Versatile Open Software Tool to Quantify Cardiomyocyte and Cardiac Muscle Contraction In Vitro and In Vivo.

PubMed

Sala, Luca; van Meer, Berend J; Tertoolen, Leon G J; Bakkers, Jeroen; Bellin, Milena; Davis, Richard P; Denning, Chris; Dieben, Michel A E; Eschenhagen, Thomas; Giacomelli, Elisa; Grandela, Catarina; Hansen, Arne; Holman, Eduard R; Jongbloed, Monique R M; Kamel, Sarah M; Koopman, Charlotte D; Lachaud, Quentin; Mannhardt, Ingra; Mol, Mervyn P H; Mosqueira, Diogo; Orlova, Valeria V; Passier, Robert; Ribeiro, Marcelo C; Saleem, Umber; Smith, Godfrey L; Burton, Francis L; Mummery, Christine L

2018-02-02

There are several methods to measure cardiomyocyte and muscle contraction, but these require customized hardware, expensive apparatus, and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming, and only specialist researchers can quantify data. Here, we describe and validate an automated, open-source software tool (MUSCLEMOTION) adaptable for use with standard laboratory and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes, and pharmacological responses. MUSCLEMOTION allowed rapid and easy measurement of movement from high-speed movies in (1) 1-dimensional in vitro models, such as isolated adult and human pluripotent stem cell-derived cardiomyocytes; (2) 2-dimensional in vitro models, such as beating cardiomyocyte monolayers or small clusters of human pluripotent stem cell-derived cardiomyocytes; (3) 3-dimensional multicellular in vitro or in vivo contractile tissues, such as cardiac "organoids," engineered heart tissues, and zebrafish and human hearts. MUSCLEMOTION was effective under different recording conditions (bright-field microscopy with simultaneous patch-clamp recording, phase contrast microscopy, and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement, such as optical flow, post deflection, edge-detection systems, or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. Using a single open-source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell, animal, and human models. © 2017 The Authors.

Metabolism of captopril carboxyl ester derivatives for percutaneous absorption.

PubMed

Gullick, Darren R; Ingram, Matthew J; Pugh, W John; Cox, Paul A; Gard, Paul; Smart, John D; Moss, Gary P

2009-02-01

To determine the metabolism of captopril n-carboxyl derivatives and how this may impact on their use as transdermal prodrugs. The pharmacological activity of the ester derivatives was also characterised in order to compare the angiotensin converting enzyme inhibitory potency of the derivatives compared with the parent drug, captopril. The metabolism rates of the ester derivatives were determined in vitro (using porcine liver esterase and porcine ear skin) and in silico (using molecular modelling to investigate the potential to predict metabolism). Relatively slow pseudo first-order metabolism of the prodrugs was observed, with the ethyl ester displaying the highest rate of metabolism. A strong relationship was established between in-vitro methods, while in-silico methods support the use of in-vitro methods and highlight the potential of in-silico techniques to predict metabolism. All the prodrugs behaved as angiotensin converting enzyme inhibitors, with the methyl ester displaying optimum inhibition. In-vitro porcine liver esterase metabolism rates inform in-vitro skin rates well, and in-silico interaction energies relate well to both. Thus, in-silico methods may be developed that include interaction energies to predict metabolism rates.

Modified methods for growing 3-D skin equivalents: an update.

PubMed

Lamb, Rebecca; Ambler, Carrie A

2014-01-01

Artificial epidermis can be reconstituted in vitro by seeding primary epidermal cells (keratinocytes) onto a supportive substrate and then growing the developing skin equivalent at the air-liquid interface. In vitro skin models are widely used to study skin biology and for industrial drug and cosmetic testing. Here, we describe updated methods for growing 3-dimensional skin equivalents using de-vitalized, de-epidermalized dermis (DED) substrates including methods for DED substrate preparation, cell seeding, growth conditions, and fixation procedures.

Micro pore arrays in free standing cyclic olefin copolymer membranes: fabrication and surface functionalization strategies for in-vitro barrier tissue models

NASA Astrophysics Data System (ADS)

Gel, M.; Kandasamy, S.; Cartledge, K.; Be, C. L.; Haylock, D.

2013-12-01

In recent years there has been growing interest in micro engineered in-vitro models of tissues and organs. These models are designed to mimic the in-vivo like physiological conditions with a goal to study human physiology in an organ-specific context or to develop in-vitro disease models. One of the challenges in the development of these models is the formation of barrier tissues in which the permeability is controlled locally by the tissues cultured at the interface. In-vitro models of barrier tissues are typically created by generating a monolayer of cells grown on thin porous membranes. This paper reports a robust preparation method for free standing porous cyclic olefin copolymer (COC) membranes. We also demonstrate that gelatin coated membranes facilitate formation of highly confluent monolayer of HUVECs. Membranes with thickness in the range of 2-3 um incorporating micro pores with diameter approximately 20 um were fabricated and integrated with microfluidic channels. The performance of the device was demonstrated with a model system mimicking the endothelial barrier in bone marrow sinusoids.

High throughput ADME screening: practical considerations, impact on the portfolio and enabler of in silico ADME models.

PubMed

Hop, Cornelis E C A; Cole, Mark J; Davidson, Ralph E; Duignan, David B; Federico, James; Janiszewski, John S; Jenkins, Kelly; Krueger, Suzanne; Lebowitz, Rebecca; Liston, Theodore E; Mitchell, Walter; Snyder, Mark; Steyn, Stefan J; Soglia, John R; Taylor, Christine; Troutman, Matt D; Umland, John; West, Michael; Whalen, Kevin M; Zelesky, Veronica; Zhao, Sabrina X

2008-11-01

Evaluation and optimization of drug metabolism and pharmacokinetic data plays an important role in drug discovery and development and several reliable in vitro ADME models are available. Recently higher throughput in vitro ADME screening facilities have been established in order to be able to evaluate an appreciable fraction of synthesized compounds. The ADME screening process can be dissected in five distinct steps: (1) plate management of compounds in need of in vitro ADME data, (2) optimization of the MS/MS method for the compounds, (3) in vitro ADME experiments and sample clean up, (4) collection and reduction of the raw LC-MS/MS data and (5) archival of the processed ADME data. All steps will be described in detail and the value of the data on drug discovery projects will be discussed as well. Finally, in vitro ADME screening can generate large quantities of data obtained under identical conditions to allow building of reliable in silico models.

Toxicokinetic Triage for Environmental Chemicals | Science ...

EPA Pesticide Factsheets

Toxicokinetic (TK) models are essential for linking administered doses to blood and tissue concentrations. In vitro-to-in vivo extrapolation (IVIVE) methods have been developed to determine TK from limited in vitro measurements and chemical structure-based property predictions, providing a less resource–intensive alternative to traditional in vivo TK approaches. High throughput TK (HTTK) methods use IVIVE to estimate doses that produce steady-state plasma concentrations equivalent to those producing biological activity in in vitro screening studies (e.g., ToxCast). In this study, the domain of applicability and assumptions of HTTK approaches were evaluated using both in vivo data and simulation analysis. Based on in vivo data for 87 chemicals, specific properties (e.g., in vitro HTTK data, physico-chemical descriptors, chemical structure, and predicted transporter affinities) were identified that correlate with poor HTTK predictive ability. For 350 xenobiotics with literature HTTK data, we then differentiated those xenobiotics for which HTTK approaches are likely to be sufficient, from those that may require additional data. For 272 chemicals we also developed a HT physiologically-based TK (HTPBTK) model that requires somewhat greater information than a steady-state model, but allows non-steady state dynamics and can predict chemical concentration time-courses for a variety of exposure scenarios, tissues, and species. We used this HTPBTK model to show that the

QUANTITATIVE IN VITRO MEASUREMENT OF CELLULAR PROCESSES CRITICAL TO THE DEVELOPMENT OF NEURAL CONNECTIVITY USING HCA.

EPA Science Inventory

New methods are needed to screen thousands of environmental chemicals for toxicity, including developmental neurotoxicity. In vitro, cell-based assays that model key cellular events have been proposed for high throughput screening of chemicals for developmental neurotoxicity. Whi...

3D bioprinting: improving in vitro models of metastasis with heterogeneous tumor microenvironments

PubMed Central

Albritton, Jacob L.

2017-01-01

ABSTRACT Even with many advances in treatment over the past decades, cancer still remains a leading cause of death worldwide. Despite the recognized relationship between metastasis and increased mortality rate, surprisingly little is known about the exact mechanism of metastatic progression. Currently available in vitro models cannot replicate the three-dimensionality and heterogeneity of the tumor microenvironment sufficiently to recapitulate many of the known characteristics of tumors in vivo. Our understanding of metastatic progression would thus be boosted by the development of in vitro models that could more completely capture the salient features of cancer biology. Bioengineering groups have been working for over two decades to create in vitro microenvironments for application in regenerative medicine and tissue engineering. Over this time, advances in 3D printing technology and biomaterials research have jointly led to the creation of 3D bioprinting, which has improved our ability to develop in vitro models with complexity approaching that of the in vivo tumor microenvironment. In this Review, we give an overview of 3D bioprinting methods developed for tissue engineering, which can be directly applied to constructing in vitro models of heterogeneous tumor microenvironments. We discuss considerations and limitations associated with 3D printing and highlight how these advances could be harnessed to better model metastasis and potentially guide the development of anti-cancer strategies. PMID:28067628

3D bioprinting: improving in vitro models of metastasis with heterogeneous tumor microenvironments.

PubMed

Albritton, Jacob L; Miller, Jordan S

2017-01-01

Even with many advances in treatment over the past decades, cancer still remains a leading cause of death worldwide. Despite the recognized relationship between metastasis and increased mortality rate, surprisingly little is known about the exact mechanism of metastatic progression. Currently available in vitro models cannot replicate the three-dimensionality and heterogeneity of the tumor microenvironment sufficiently to recapitulate many of the known characteristics of tumors in vivo Our understanding of metastatic progression would thus be boosted by the development of in vitro models that could more completely capture the salient features of cancer biology. Bioengineering groups have been working for over two decades to create in vitro microenvironments for application in regenerative medicine and tissue engineering. Over this time, advances in 3D printing technology and biomaterials research have jointly led to the creation of 3D bioprinting, which has improved our ability to develop in vitro models with complexity approaching that of the in vivo tumor microenvironment. In this Review, we give an overview of 3D bioprinting methods developed for tissue engineering, which can be directly applied to constructing in vitro models of heterogeneous tumor microenvironments. We discuss considerations and limitations associated with 3D printing and highlight how these advances could be harnessed to better model metastasis and potentially guide the development of anti-cancer strategies. © 2017. Published by The Company of Biologists Ltd.

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier

PubMed Central

2013-01-01

Background Modelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies. Methods Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting. Results Using a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS endothelial cell types can be obtained, and these can be passaged onto large numbers of cell culture inserts for in vitro permeability studies. The passaged brain and spinal cord endothelial cells are pure and express endothelial markers, tight junction proteins and intracellular transport machinery. Further, both models exhibit tight, functional barrier characteristics that are discriminating against large and small molecules in permeability assays and show functional expression of the pharmaceutically important P-gp efflux transporter. Conclusions Our techniques allow the provision of high yields of robust sister cultures of endothelial cells that accurately model the blood-CNS barriers in vitro. These models are ideally suited for use in studying the biology of the blood-brain barrier and blood-spinal cord barrier in vitro and for pre-clinical drug discovery. PMID:23773766

A reproducible accelerated in vitro release testing method for PLGA microspheres.

PubMed

Shen, Jie; Lee, Kyulim; Choi, Stephanie; Qu, Wen; Wang, Yan; Burgess, Diane J

2016-02-10

The objective of the present study was to develop a discriminatory and reproducible accelerated in vitro release method for long-acting PLGA microspheres with inner structure/porosity differences. Risperidone was chosen as a model drug. Qualitatively and quantitatively equivalent PLGA microspheres with different inner structure/porosity were obtained using different manufacturing processes. Physicochemical properties as well as degradation profiles of the prepared microspheres were investigated. Furthermore, in vitro release testing of the prepared risperidone microspheres was performed using the most common in vitro release methods (i.e., sample-and-separate and flow through) for this type of product. The obtained compositionally equivalent risperidone microspheres had similar drug loading but different inner structure/porosity. When microsphere particle size appeared similar, porous risperidone microspheres showed faster microsphere degradation and drug release compared with less porous microspheres. Both in vitro release methods investigated were able to differentiate risperidone microsphere formulations with differences in porosity under real-time (37 °C) and accelerated (45 °C) testing conditions. Notably, only the accelerated USP apparatus 4 method showed good reproducibility for highly porous risperidone microspheres. These results indicated that the accelerated USP apparatus 4 method is an appropriate fast quality control tool for long-acting PLGA microspheres (even with porous structures). Copyright © 2015 Elsevier B.V. All rights reserved.

Stress Distribution in Single Dental Implant System: Three-Dimensional Finite Element Analysis Based on an In Vitro Experimental Model.

PubMed

Rezende, Carlos Eduardo Edwards; Chase-Diaz, Melody; Costa, Max Doria; Albarracin, Max Laurent; Paschoeto, Gabriela; Sousa, Edson Antonio Capello; Rubo, José Henrique; Borges, Ana Flávia Sanches

2015-10-01

This study aimed to analyze the stress distribution in single implant system and to evaluate the compatibility of an in vitro model with finite element (FE) model. The in vitro model consisted of Brånemark implant; multiunit set abutment of 5 mm height; metal-ceramic screw-retained crown, and polyurethane simulating the bone. Deformations were recorded in the peri-implant region in the mesial and distal aspects, after an axial 300 N load application at the center of the occlusal aspect of the crown, using strain gauges. This in vitro model was scanned with micro CT to design a three-dimensional FE model and the strains in the peri-implant bone region were registered to check the compatibility between both models. The FE model was used to evaluate stress distribution in different parts of the system. The values obtained from the in vitro model (20-587 με) and the finite element analysis (81-588 με) showed agreement among them. The highest stresses because of axial and oblique load, respectively were 5.83 and 40 MPa for the cortical bone, 55 and 1200 MPa for the implant, and 80 and 470 MPa for the abutment screw. The FE method proved to be effective for evaluating the deformation around single implant. Oblique loads lead to higher stress concentrations.

A new 3D reconstituted human corneal epithelium model as an alternative method for the eye irritation test.

PubMed

Jung, Kyoung-Mi; Lee, Su-Hyon; Ryu, Yang-Hwan; Jang, Won-Hee; Jung, Haeng-Sun; Han, Ju-Hee; Seok, Seung-Hyeok; Park, Jae-Hak; Son, Youngsook; Park, Young-Ho; Lim, Kyung-Min

2011-02-01

Many efforts are being made to develop new alternative in vitro test methods for the eye irritation test. Here we report a new reconstructed human corneal epithelial model (MCTT HCE model) prepared from primary-cultured human limbal epithelial cells as a new alternative in vitro eye irritation test method. In histological and immunohistochemical observation, MCTT HCE model displayed a morphology and biomarker expressions similar to intact human cornea. Moreover, the barrier function was well preserved as measured by high transepithelial electrical resistance, effective time-50 for Triton X-100, and corneal thickness. To employ the model as a new alternative method for eye irritation test, protocol refinement was performed and optimum assay condition was determined including treatment time, treatment volume, post-incubation time and rinsing method. Using the refined protocol, 25 reference chemicals with known eye irritation potentials were tested. With the viability cut-off value at 50%, chemicals were classified to irritant or non-irritant. When compared with GHS classification, the MCTT HCE model showed the accuracy of 88%, sensitivity of 100% and specificity of 77%. These results suggest that the MCTT HCE model might be useful as a new alternative eye irritation test method. Copyright © 2010 Elsevier Ltd. All rights reserved.

Toxicokinetic and Dosimetry Modeling Tools for Exposure Reconstruction: US EPA's Rapid Exposure and Dosimetry (RED) Project

EPA Science Inventory

New technologies and in vitro testing approaches have been valuable additions to risk assessments that have historically relied solely on in vivo test results. Compared to in vivo methods, in vitro high throughput screening (HTS) assays are less expensive, faster and can provide ...

ASSESSMENT OF CHEMICAL EFFECTS ON NEURITE OUTGROWTH, NEURONAL POLARIZATION AND SYNAPTOGENESIS IN RAT CORTICAL NEURONS USING HIGH CONTENT IMAGE ANALYSIS

EPA Science Inventory

There is a need for efficient, cost-effective methods for screening and prioritization of potential developmental neurotoxicants. One approach uses in vitro cell culture models that can recapitulate the critical processes of nervous system development. In vitro, primary cultures ...

Advances in In Vitro and In Silico Tools for Toxicokinetic Dose Modeling and Predictive Toxicology (WC10)

EPA Science Inventory

Recent advances in vitro assays, in silico tools, and systems biology approaches provide opportunities for refined mechanistic understanding for chemical safety assessment that will ultimately lead to reduced reliance on animal-based methods. With the U.S. commercial chemical lan...

Validation of an In Vitro Sun Protection Factor (SPF) method in blinded ring-testing.

PubMed

Pissavini, M; Tricaud, C; Wiener, G; Lauer, A; Contier, M; Kolbe, L; Trullás Cabanas, C; Boyer, F; Nollent, V; Meredith, E; Dietrich, E; Matts, P J

2018-04-20

The objective of this work was to investigate the utility of a new in vitro SPF test method in blinded ring-testing, against new ISO acceptance criteria. 24 blinded, commercial, emulsion-type, primary sunscreen products, covering the full range of labelled SPF in Europe (SPF6 - 50+), were tested by 3 test institutes using the current ISO24444:2010 In Vivo SPF Test Method and simultaneously by 3 separate test laboratories using a new candidate in vitro SPF test method, developed under the leadership of Cosmetics Europe (CE). The resulting relationship between in vitro SPF and in vivo SPF values was then compared with acceptance criteria developed recently by the International Standards (ISO) TC217 / WG7 Sun Protection Test Methods Working Group. Analysis of the mean inter-laboratory in vitro and mean inter-institute in vivo SPF values revealed a strong correlation between in vitro and in vivo values, with a Pearson correlation coefficient of r 2 =0.88 (p<0.0001), a slope of 1.01 and a non-significant intercept (-1.48; p=0.62). When these data were compared to the new ISO WG7 acceptance criteria, method bias was found to be extremely low and over 95% of the coupled data lay within the model "funnel" (defined by upper and lower confidence intervals). In conclusion, the results of blinded ring testing and comparison to new ISO WG7 acceptance criteria indicate that a new in vitro SPF test method meets (and exceeds) these minimum criteria and is an interesting candidate for possible deployment as an industry test methodology. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Comparison of human skin irritation patch test data with in vitro skin irritation assays and animal data.

PubMed

Jírová, Dagmar; Basketter, David; Liebsch, Manfred; Bendová, Hana; Kejlová, Kristina; Marriott, Marie; Kandárová, Helena

2010-02-01

Efforts to replace the rabbit skin irritation test have been underway for many years, encouraged by the EU Cosmetics Directive and REACH. Recently various in vitro tests have been developed, evaluated and validated. A key difficulty in confirming the validity of in vitro methods is that animal data are scarce and of limited utility for prediction of human effects, which adversely impacts their acceptance. This study examines whether in vivo or in vitro data most accurately predicted human effects. Using the 4-hr human patch test (HPT) we examined a number of chemicals whose EU classification of skin irritancy is known to be borderline, or where in vitro methods provided conflicting results. Of the 16 chemicals classified as irritants in the rabbit, only five substances were found to be significantly irritating to human skin. Concordance of the rabbit test with the 4-hr HPT was only 56%, whereas concordance of human epidermis models with human data was 76% (EpiDerm) and 70% (EPISKIN). The results confirm observations that rabbits overpredict skin effects in humans. Therefore, when validating in vitro methods, all available information, including human data, should be taken into account before making conclusions about their predictive capacity.

Models and methods for in vitro testing of hepatic gap junctional communication.

PubMed

Maes, Michaël; Yanguas, Sara Crespo; Willebrords, Joost; Vinken, Mathieu

2015-12-25

Inherent to their pivotal roles in controlling all aspects of the liver cell life cycle, hepatocellular gap junctions are frequently disrupted upon impairment of the homeostatic balance, as occurs during liver toxicity. Hepatic gap junctions, which are mainly built up by connexin32, are specifically targeted by tumor promoters and epigenetic carcinogens. This renders inhibition of gap junction functionality a suitable indicator for the in vitro detection of nongenotoxic hepatocarcinogenicity. The establishment of a reliable liver gap junction inhibition assay for routine in vitro testing purposes requires a cellular system in which gap junctions are expressed at an in vivo-like level as well as an appropriate technique to probe gap junction activity. Both these models and methods are discussed in the current paper, thereby focusing on connexin32-based gap junctions. Copyright © 2015 Elsevier B.V. All rights reserved.

Engineering stromal-epithelial interactions in vitro for ...

EPA Pesticide Factsheets

Background: Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue function. Epithelial-mesenchymal interactions (EMIs) have been examined using mammalian models, ex vivo tissue recombination, and in vitro co-cultures. Although these approaches have elucidated signaling mechanisms underlying morphogenetic processes and adult mammalian epithelial tissue function, they are limited by the availability of human tissue, low throughput, and human developmental or physiological relevance. Objectives: Bioengineering strategies to promote EMIs using human epithelial and mesenchymal cells have enabled the development of human in vitro models of adult epidermal and glandular tissues. In this review, we describe recent bioengineered models of human epithelial tissue and organs that can instruct the design of organotypic models of human developmental processes.Methods: We reviewed current bioengineering literature and here describe how bioengineered EMIs have enabled the development of human in vitro epithelial tissue models.Discussion: Engineered models to promote EMIs have recapitulated the architecture, phenotype, and function of adult human epithelial tissue, and similar engineering principles could be used to develop models of developmental morphogenesis. We describe how bioengineering strategies including bioprinting and spheroid culture could be implemented to

Three-dimensional printing of Hela cells for cervical tumor model in vitro.

PubMed

Zhao, Yu; Yao, Rui; Ouyang, Liliang; Ding, Hongxu; Zhang, Ting; Zhang, Kaitai; Cheng, Shujun; Sun, Wei

2014-09-01

Advances in three-dimensional (3D) printing have enabled the direct assembly of cells and extracellular matrix materials to form in vitro cellular models for 3D biology, the study of disease pathogenesis and new drug discovery. In this study, we report a method of 3D printing for Hela cells and gelatin/alginate/fibrinogen hydrogels to construct in vitro cervical tumor models. Cell proliferation, matrix metalloproteinase (MMP) protein expression and chemoresistance were measured in the printed 3D cervical tumor models and compared with conventional 2D planar culture models. Over 90% cell viability was observed using the defined printing process. Comparisons of 3D and 2D results revealed that Hela cells showed a higher proliferation rate in the printed 3D environment and tended to form cellular spheroids, but formed monolayer cell sheets in 2D culture. Hela cells in 3D printed models also showed higher MMP protein expression and higher chemoresistance than those in 2D culture. These new biological characteristics from the printed 3D tumor models in vitro as well as the novel 3D cell printing technology may help the evolution of 3D cancer study.

Prevalidation of an Acute Inhalation Toxicity Test Using the EpiAirway In Vitro Human Airway Model

PubMed Central

Jackson, George R.; Maione, Anna G.; Klausner, Mitchell

2018-01-01

Abstract Introduction: Knowledge of acute inhalation toxicity potential is important for establishing safe use of chemicals and consumer products. Inhalation toxicity testing and classification procedures currently accepted within worldwide government regulatory systems rely primarily on tests conducted in animals. The goal of the current work was to develop and prevalidate a nonanimal (in vitro) test for determining acute inhalation toxicity using the EpiAirway™ in vitro human airway model as a potential alternative for currently accepted animal tests. Materials and Methods: The in vitro test method exposes EpiAirway tissues to test chemicals for 3 hours, followed by measurement of tissue viability as the test endpoint. Fifty-nine chemicals covering a broad range of toxicity classes, chemical structures, and physical properties were evaluated. The in vitro toxicity data were utilized to establish a prediction model to classify the chemicals into categories corresponding to the currently accepted Globally Harmonized System (GHS) and the Environmental Protection Agency (EPA) system. Results: The EpiAirway prediction model identified in vivo rat-based GHS Acute Inhalation Toxicity Category 1–2 and EPA Acute Inhalation Toxicity Category I–II chemicals with 100% sensitivity and specificity of 43.1% and 50.0%, for GHS and EPA acute inhalation toxicity systems, respectively. The sensitivity and specificity of the EpiAirway prediction model for identifying GHS specific target organ toxicity-single exposure (STOT-SE) Category 1 human toxicants were 75.0% and 56.5%, respectively. Corrosivity and electrophilic and oxidative reactivity appear to be the predominant mechanisms of toxicity for the most highly toxic chemicals. Conclusions: These results indicate that the EpiAirway test is a promising alternative to the currently accepted animal tests for acute inhalation toxicity. PMID:29904643

Development and Validation of a Computational Model for Androgen Receptor Activity

PubMed Central

2016-01-01

Testing thousands of chemicals to identify potential androgen receptor (AR) agonists or antagonists would cost millions of dollars and take decades to complete using current validated methods. High-throughput in vitro screening (HTS) and computational toxicology approaches can more rapidly and inexpensively identify potential androgen-active chemicals. We integrated 11 HTS ToxCast/Tox21 in vitro assays into a computational network model to distinguish true AR pathway activity from technology-specific assay interference. The in vitro HTS assays probed perturbations of the AR pathway at multiple points (receptor binding, coregulator recruitment, gene transcription, and protein production) and multiple cell types. Confirmatory in vitro antagonist assay data and cytotoxicity information were used as additional flags for potential nonspecific activity. Validating such alternative testing strategies requires high-quality reference data. We compiled 158 putative androgen-active and -inactive chemicals from a combination of international test method validation efforts and semiautomated systematic literature reviews. Detailed in vitro assay information and results were compiled into a single database using a standardized ontology. Reference chemical concentrations that activated or inhibited AR pathway activity were identified to establish a range of potencies with reproducible reference chemical results. Comparison with existing Tier 1 AR binding data from the U.S. EPA Endocrine Disruptor Screening Program revealed that the model identified binders at relevant test concentrations (<100 μM) and was more sensitive to antagonist activity. The AR pathway model based on the ToxCast/Tox21 assays had balanced accuracies of 95.2% for agonist (n = 29) and 97.5% for antagonist (n = 28) reference chemicals. Out of 1855 chemicals screened in the AR pathway model, 220 chemicals demonstrated AR agonist or antagonist activity and an additional 174 chemicals were predicted to have potential weak AR pathway activity. PMID:27933809

Prevalidation of an Acute Inhalation Toxicity Test Using the EpiAirway In Vitro Human Airway Model.

PubMed

Jackson, George R; Maione, Anna G; Klausner, Mitchell; Hayden, Patrick J

2018-06-01

Introduction: Knowledge of acute inhalation toxicity potential is important for establishing safe use of chemicals and consumer products. Inhalation toxicity testing and classification procedures currently accepted within worldwide government regulatory systems rely primarily on tests conducted in animals. The goal of the current work was to develop and prevalidate a nonanimal ( in vitro ) test for determining acute inhalation toxicity using the EpiAirway™ in vitro human airway model as a potential alternative for currently accepted animal tests. Materials and Methods: The in vitro test method exposes EpiAirway tissues to test chemicals for 3 hours, followed by measurement of tissue viability as the test endpoint. Fifty-nine chemicals covering a broad range of toxicity classes, chemical structures, and physical properties were evaluated. The in vitro toxicity data were utilized to establish a prediction model to classify the chemicals into categories corresponding to the currently accepted Globally Harmonized System (GHS) and the Environmental Protection Agency (EPA) system. Results: The EpiAirway prediction model identified in vivo rat-based GHS Acute Inhalation Toxicity Category 1-2 and EPA Acute Inhalation Toxicity Category I-II chemicals with 100% sensitivity and specificity of 43.1% and 50.0%, for GHS and EPA acute inhalation toxicity systems, respectively. The sensitivity and specificity of the EpiAirway prediction model for identifying GHS specific target organ toxicity-single exposure (STOT-SE) Category 1 human toxicants were 75.0% and 56.5%, respectively. Corrosivity and electrophilic and oxidative reactivity appear to be the predominant mechanisms of toxicity for the most highly toxic chemicals. Conclusions: These results indicate that the EpiAirway test is a promising alternative to the currently accepted animal tests for acute inhalation toxicity.

Integration of QSAR and in vitro toxicology.

PubMed Central

Barratt, M D

1998-01-01

The principles of quantitative structure-activity relationships (QSAR) are based on the premise that the properties of a chemical are implicit in its molecular structure. Therefore, if a mechanistic hypothesis can be proposed linking a group of related chemicals with a particular toxic end point, the hypothesis can be used to define relevant parameters to establish a QSAR. Ways in which QSAR and in vitro toxicology can complement each other in development of alternatives to live animal experiments are described and illustrated by examples from acute toxicological end points. Integration of QSAR and in vitro methods is examined in the context of assessing mechanistic competence and improving the design of in vitro assays and the development of prediction models. The nature of biological variability is explored together with its implications for the selection of sets of chemicals for test development, optimization, and validation. Methods are described to support the use of data from in vivo tests that do not meet today's stringent requirements of acceptability. Integration of QSAR and in vitro methods into strategic approaches for the replacement, reduction, and refinement of the use of animals is described with examples. PMID:9599692

Virtual Liver: integrating in vitro and in vivo data to predict chemical-induced toxicity

EPA Science Inventory

It is difficult to assess the health impact of long-term exposure to low levels of contaminants from animal studies. Current methods for testing the toxicity of a single chemical can cost millions of dollars, take up to two years and sacrifice thousands of animals. In vitro model...

Evaluation of Pharmacokinetic Assumptions Using a 443 ...

EPA Pesticide Factsheets

With the increasing availability of high-throughput and in vitro data for untested chemicals, there is a need for pharmacokinetic (PK) models for in vitro to in vivo extrapolation (IVIVE). Though some PBPK models have been created for individual compounds using in vivo data, we are now able to rapidly parameterize generic PBPK models using in vitro data to allow IVIVE for chemicals tested for bioactivity via high-throughput screening. However, these new models are expected to have limited accuracy due to their simplicity and generalization of assumptions. We evaluated the assumptions and performance of a generic PBPK model (R package “httk”) parameterized by a library of in vitro PK data for 443 chemicals. We evaluate and calibrate Schmitt’s method by comparing the predicted volume of distribution (Vd) and tissue partition coefficients to in vivo measurements. The partition coefficients are initially over predicted, likely due to overestimation of partitioning into phospholipids in tissues and the lack of lipid partitioning in the in vitro measurements of the fraction unbound in plasma. Correcting for phospholipids and plasma binding improved the predictive ability (R2 to 0.52 for partition coefficients and 0.32 for Vd). We lacked enough data to evaluate the accuracy of changing the model structure to include tissue blood volumes and/or separate compartments for richly/poorly perfused tissues, therefore we evaluated the impact of these changes on model

Identification of candidate reference chemicals for in vitro steroidogenesis assays.

PubMed

Pinto, Caroline Lucia; Markey, Kristan; Dix, David; Browne, Patience

2018-03-01

The Endocrine Disruptor Screening Program (EDSP) is transitioning from traditional testing methods to integrating ToxCast/Tox21 in vitro high-throughput screening assays for identifying chemicals with endocrine bioactivity. The ToxCast high-throughput H295R steroidogenesis assay may potentially replace the low-throughput assays currently used in the EDSP Tier 1 battery to detect chemicals that alter the synthesis of androgens and estrogens. Herein, we describe an approach for identifying in vitro candidate reference chemicals that affect the production of androgens and estrogens in models of steroidogenesis. Candidate reference chemicals were identified from a review of H295R and gonad-derived in vitro assays used in methods validation and published in the scientific literature. A total of 29 chemicals affecting androgen and estrogen levels satisfied all criteria for positive reference chemicals, while an additional set of 21 and 15 chemicals partially fulfilled criteria for positive reference chemicals for androgens and estrogens, respectively. The identified chemicals included pesticides, pharmaceuticals, industrial and naturally-occurring chemicals with the capability to increase or decrease the levels of the sex hormones in vitro. Additionally, 14 and 15 compounds were identified as potential negative reference chemicals for effects on androgens and estrogens, respectively. These candidate reference chemicals will be informative for performance-based validation of in vitro steroidogenesis models. Copyright © 2017. Published by Elsevier Ltd.

Air-liquid interface exposure to aerosols of poorly soluble nanomaterials induces different biological activation levels compared to exposure to suspensions.

PubMed

Loret, Thomas; Peyret, Emmanuel; Dubreuil, Marielle; Aguerre-Chariol, Olivier; Bressot, Christophe; le Bihan, Olivier; Amodeo, Tanguy; Trouiller, Bénédicte; Braun, Anne; Egles, Christophe; Lacroix, Ghislaine

2016-11-03

Recently, much progress has been made to develop more physiologic in vitro models of the respiratory system and improve in vitro simulation of particle exposure through inhalation. Nevertheless, the field of nanotoxicology still suffers from a lack of relevant in vitro models and exposure methods to predict accurately the effects observed in vivo, especially after respiratory exposure. In this context, the aim of our study was to evaluate if exposing pulmonary cells at the air-liquid interface to aerosols of inhalable and poorly soluble nanomaterials generates different toxicity patterns and/or biological activation levels compared to classic submerged exposures to suspensions. Three nano-TiO 2 and one nano-CeO 2 were used. An exposure system was set up using VitroCell® devices to expose pulmonary cells at the air-liquid interface to aerosols. A549 alveolar cells in monocultures or in co-cultures with THP-1 macrophages were exposed to aerosols in inserts or to suspensions in inserts and in plates. Submerged exposures in inserts were performed, using similar culture conditions and exposure kinetics to the air-liquid interface, to provide accurate comparisons between the methods. Exposure in plates using classical culture and exposure conditions was performed to provide comparable results with classical submerged exposure studies. The biological activity of the cells (inflammation, cell viability, oxidative stress) was assessed at 24 h and comparisons of the nanomaterial toxicities between exposure methods were performed. Deposited doses of nanomaterials achieved using our aerosol exposure system were sufficient to observe adverse effects. Co-cultures were more sensitive than monocultures and biological responses were usually observed at lower doses at the air-liquid interface than in submerged conditions. Nevertheless, the general ranking of the nanomaterials according to their toxicity was similar across the different exposure methods used. We showed that exposure of cells at the air-liquid interface represents a valid and sensitive method to assess the toxicity of several poorly soluble nanomaterials. We underlined the importance of the cellular model used and offer the possibility to deal with low deposition doses by using more sensitive and physiologic cellular models. This brings perspectives towards the use of relevant in vitro methods of exposure to assess nanomaterial toxicity.

Methods and options in vitro dialyzability; benefits and limitations.

PubMed

Sandberg, Ann-Sofie

2005-11-01

In vitro dialyzability methods involve a two-step digestion process simulating the gastric and intestinal phase, and dialysis through a semi-permeable membrane with a selected molecular weight cut-off. Dialyzable iron/zinc is used as an estimation of available mineral. Final pH adjustment and use of a strict time schedule were found to be critical factors for standardization. In addition the selected cut-off of the dialysis membrane and the method used for iron and zinc determination influence the results. For screening purposes, simple solubility or dialyzability methods seem preferable to the more sophisticated computer-controlled gastrointestinal model. This is likely more valuable in studies of different transit times and sites of dialyzability. In vitro solubility/dialyzability methods correlate in most cases with human absorption studies in ranking iron and zinc availability from different meals. Exceptions may be that effects of milk, certain proteins, tea, and organic acids cannot be predicted. The dialyzability methods exclude iron bound to large molecules, which in some cases is available and include iron bound to small molecules, which is not always available. In vitro experiments based on solubility/dialyzability are tools to understand factors that may affect subsequent mineral absorption.

Approach for extrapolating in vitro metabolism data to refine bioconcentration factor estimates.

PubMed

Cowan-Ellsberry, Christina E; Dyer, Scott D; Erhardt, Susan; Bernhard, Mary Jo; Roe, Amy L; Dowty, Martin E; Weisbrod, Annie V

2008-02-01

National and international chemical management programs are assessing thousands of chemicals for their persistence, bioaccumulative and environmental toxic properties; however, data for evaluating the bioaccumulation potential for fish are limited. Computer based models that account for the uptake and elimination processes that contribute to bioaccumulation may help to meet the need for reliable estimates. One critical elimination process of chemicals is metabolic transformation. It has been suggested that in vitro metabolic transformation tests using fish liver hepatocytes or S9 fractions can provide rapid and cost-effective measurements of fish metabolic potential, which could be used to refine bioconcentration factor (BCF) computer model estimates. Therefore, recent activity has focused on developing in vitro methods to measure metabolic transformation in cellular and subcellular fish liver fractions. A method to extrapolate in vitro test data to the whole body metabolic transformation rates is presented that could be used to refine BCF computer model estimates. This extrapolation approach is based on concepts used to determine the fate and distribution of drugs within the human body which have successfully supported the development of new pharmaceuticals for years. In addition, this approach has already been applied in physiologically-based toxicokinetic models for fish. The validity of the in vitro to in vivo extrapolation is illustrated using the rate of loss of parent chemical measured in two independent in vitro test systems: (1) subcellular enzymatic test using the trout liver S9 fraction, and (2) primary hepatocytes isolated from the common carp. The test chemicals evaluated have high quality in vivo BCF values and a range of logK(ow) from 3.5 to 6.7. The results show very good agreement between the measured BCF and estimated BCF values when the extrapolated whole body metabolism rates are included, thus suggesting that in vitro biotransformation data could effectively be used to reduce in vivo BCF testing and refine BCF model estimates. However, additional fish physiological data for parameterization and validation for a wider range of chemicals are needed.

Modelling human skull growth: a validated computational model

PubMed Central

Marghoub, Arsalan; Johnson, David; Khonsari, Roman H.; Fagan, Michael J.; Moazen, Mehran

2017-01-01

During the first year of life, the brain grows rapidly and the neurocranium increases to about 65% of its adult size. Our understanding of the relationship between the biomechanical forces, especially from the growing brain, the craniofacial soft tissue structures and the individual bone plates of the skull vault is still limited. This basic knowledge could help in the future planning of craniofacial surgical operations. The aim of this study was to develop a validated computational model of skull growth, based on the finite-element (FE) method, to help understand the biomechanics of skull growth. To do this, a two-step validation study was carried out. First, an in vitro physical three-dimensional printed model and an in silico FE model were created from the same micro-CT scan of an infant skull and loaded with forces from the growing brain from zero to two months of age. The results from the in vitro model validated the FE model before it was further developed to expand from 0 to 12 months of age. This second FE model was compared directly with in vivo clinical CT scans of infants without craniofacial conditions (n = 56). The various models were compared in terms of predicted skull width, length and circumference, while the overall shape was quantified using three-dimensional distance plots. Statistical analysis yielded no significant differences between the male skull models. All size measurements from the FE model versus the in vitro physical model were within 5%, with one exception showing a 7.6% difference. The FE model and in vivo data also correlated well, with the largest percentage difference in size being 8.3%. Overall, the FE model results matched well with both the in vitro and in vivo data. With further development and model refinement, this modelling method could be used to assist in preoperative planning of craniofacial surgery procedures and could help to reduce reoperation rates. PMID:28566514

Modelling human skull growth: a validated computational model.

PubMed

Libby, Joseph; Marghoub, Arsalan; Johnson, David; Khonsari, Roman H; Fagan, Michael J; Moazen, Mehran

2017-05-01

During the first year of life, the brain grows rapidly and the neurocranium increases to about 65% of its adult size. Our understanding of the relationship between the biomechanical forces, especially from the growing brain, the craniofacial soft tissue structures and the individual bone plates of the skull vault is still limited. This basic knowledge could help in the future planning of craniofacial surgical operations. The aim of this study was to develop a validated computational model of skull growth, based on the finite-element (FE) method, to help understand the biomechanics of skull growth. To do this, a two-step validation study was carried out. First, an in vitro physical three-dimensional printed model and an in silico FE model were created from the same micro-CT scan of an infant skull and loaded with forces from the growing brain from zero to two months of age. The results from the in vitro model validated the FE model before it was further developed to expand from 0 to 12 months of age. This second FE model was compared directly with in vivo clinical CT scans of infants without craniofacial conditions ( n = 56). The various models were compared in terms of predicted skull width, length and circumference, while the overall shape was quantified using three-dimensional distance plots. Statistical analysis yielded no significant differences between the male skull models. All size measurements from the FE model versus the in vitro physical model were within 5%, with one exception showing a 7.6% difference. The FE model and in vivo data also correlated well, with the largest percentage difference in size being 8.3%. Overall, the FE model results matched well with both the in vitro and in vivo data. With further development and model refinement, this modelling method could be used to assist in preoperative planning of craniofacial surgery procedures and could help to reduce reoperation rates. © 2017 The Author(s).

Lacking applicability of in vitro eye irritation methods to identify seriously eye irritating agrochemical formulations: Results of bovine cornea opacity and permeability assay, isolated chicken eye test and the EpiOcular™ ET-50 method to classify according to UN GHS.

PubMed

Kolle, Susanne N; Van Cott, Andrew; van Ravenzwaay, Bennard; Landsiedel, Robert

2017-04-01

In vitro methods have gained regulatory acceptance for the prediction of serious eye damage (UN GHS Cat 1). However, the majority of in vitro methods do not state whether they are applicable to agrochemical formulations. This manuscript presents a study of up to 27 agrochemical formulations tested in three in vitro assays (three versions of the bovine corneal opacity and permeability test (BCOP, OECD TG 437) assay, the isolated chicken eye test (ICE, OECD TG 438) and the EpiOcular™ ET-50 assay). The results were compared with already-available in vivo data. In the BCOP only one of the four, one of five in the ICE and six of eleven tested formulations in the EpiOcular™ ET-50 Neat Protocol resulted in the correct UN GHS Cat 1 prediction. Overpredictions occurred in all assays. These data indicate a lack of applicability of the three in vitro methods to reliably predict UN GHS Cat 1 of agrochemical formulations. In order to ensure animal-free identification of seriously eye damaging agrochemical formulations testing protocols and/or prediction models need to be modified or classification rules should be tailored to in vitro testing rather than using in vivo Draize data as a standard. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Evaluating the use of optical coherence tomography for the detection of epithelial cancers in vitro

NASA Astrophysics Data System (ADS)

Smith, Louise E.; Hearnden, Vanessa; Lu, Zenghai; Smallwood, Rod; Hunter, Keith D.; Matcher, Stephen J.; Thornhill, Martin H.; Murdoch, Craig; MacNeil, Sheila

2011-11-01

Optical coherence tomography (OCT) is a noninvasive imaging methodology that is able to image tissue to depths of over 1 mm. Many epithelial conditions, such as melanoma and oral cancers, require an invasive biopsy for diagnosis. A noninvasive, real-time, point of care method of imaging depth-resolved epithelial structure could greatly improve early diagnosis and long-term monitoring in patients. Here, we have used tissue-engineered (TE) models of normal skin and oral mucosa to generate models of melanoma and oral cancer. We have used these to determine the ability of OCT to image epithelial differences in vitro. We report that while in vivo OCT gives reasonable depth information for both skin and oral mucosa, in vitro the information provided is less detailed but still useful. OCT can provide reassurance on the development of TE models of skin and oral mucosa as they develop in vitro. OCT was able to detect the gross alteration in the epithelium of skin and mucosal models generated with malignant cell lines but was less able to detect alteration in the epithelium of TE models that mimicked oral dysplasia or, in models where tumor cells had penetrated into the dermis.

[Application of numerical convolution in in vivo/in vitro correlation research].

PubMed

Yue, Peng

2009-01-01

This paper introduced the conception and principle of in vivo/in vitro correlation (IVIVC) and convolution/deconvolution methods, and elucidated in details the convolution strategy and method for calculating the in vivo absorption performance of the pharmaceutics according to the their pharmacokinetic data in Excel, then put the results forward to IVIVC research. Firstly, the pharmacokinetic data ware fitted by mathematical software to make up the lost points. Secondly, the parameters of the optimal fitted input function were defined by trail-and-error method according to the convolution principle in Excel under the hypothesis that all the input functions fit the Weibull functions. Finally, the IVIVC between in vivo input function and the in vitro dissolution was studied. In the examples, not only the application of this method was demonstrated in details but also its simplicity and effectiveness were proved by comparing with the compartment model method and deconvolution method. It showed to be a powerful tool for IVIVC research.

Validation of an in vitro model for predicting rumen and total-tract fiber digestibility in dairy cows fed corn silages with different in vitro neutral detergent fiber digestibilities at 2 levels of dry matter intake.

PubMed

Lopes, F; Cook, D E; Combs, D K

2015-01-01

An in vivo study was performed to validate an in vitro procedure that predicts rate of fiber digestion and total-tract neutral detergent fiber digestibility (TTNDFD). Two corn silages that differed in fiber digestibility were used in this trial. The corn silage with lower fiber digestibility (LFDCS) had the TTNDFD prediction of 36.0% of total NDF, whereas TTNDFD for the corn silage with higher fiber digestibility (HFDCS) was 44.9% of total neutral detergent fiber (NDF). Two diets (1 with LFDCS and 1 with HFDCS) were formulated and analyzed using the in vitro assay to predict the TTNDFD and rumen potentially digestible NDF (pdNDF) digestion rate. Similar diets were fed to 8 ruminally cannulated, multiparous, high-producing dairy cows in 2 replicated 4×4 Latin squares with 21-d periods. A 2×2 factorial arrangement of treatments was used with main effects of intake (restricted to approximately 90% of ad libitum intake vs. ad libitum) and corn silage of different fiber digestibility. Treatments were restricted and ad libitum LFDCS as well as restricted and ad libitum HFDCS. The input and output values predicted from the in vitro model were compared with in vivo measurements. The pdNDF intake predicted by the in vitro model was similar to pdNDF intake observed in vivo. Also, the pdNDF digestion rate predicted in vitro was similar to what was observed in vivo. The in vitro method predicted TTNDFD of 50.2% for HFDCS and 42.9% for LFDCS as a percentage of total NDF in the diets, whereas the in vivo measurements of TTNDFD averaged 50.3 and 48.6% of total NDF for the HFDCS and LFDCS diets, respectively. The in vitro TTNDFD assay predicted total-tract NDF digestibility of HFDCS diets similar to the digestibility observed in vivo, but for LFDCS diets the assay underestimated the digestibility compared with in vivo. When the in vitro and in vivo measurements were compared without intake effect (ad libitum and restricted) considering only diet effect of silage fiber digestibility (HFDCS and LFDCS), no differences were observed between methods. These values suggest that our in vitro TTNDFD model could be used to predicted rate of fiber digestion and NDF digestibility for dairy cattle. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Assessment of human epidermal model LabCyte EPI-MODEL for in vitro skin irritation testing according to European Centre for the Validation of Alternative Methods (ECVAM)-validated protocol.

PubMed

Katoh, Masakazu; Hamajima, Fumiyasu; Ogasawara, Takahiro; Hata, Ken-Ichiro

2009-06-01

A validation study of an in vitro skin irritation testing method using a reconstructed human skin model has been conducted by the European Centre for the Validation of Alternative Methods (ECVAM), and a protocol using EpiSkin (SkinEthic, France) has been approved. The structural and performance criteria of skin models for testing are defined in the ECVAM Performance Standards announced along with the approval. We have performed several evaluations of the new reconstructed human epidermal model LabCyte EPI-MODEL, and confirmed that it is applicable to skin irritation testing as defined in the ECVAM Performance Standards. We selected 19 materials (nine irritants and ten non-irritants) available in Japan as test chemicals among the 20 reference chemicals described in the ECVAM Performance Standard. A test chemical was applied to the surface of the LabCyte EPI-MODEL for 15 min, after which it was completely removed and the model then post-incubated for 42 hr. Cell v iability was measured by MTT assay and skin irritancy of the test chemical evaluated. In addition, interleukin-1 alpha (IL-1alpha) concentration in the culture supernatant after post-incubation was measured to provide a complementary evaluation of skin irritation. Evaluation of the 19 test chemicals resulted in 79% accuracy, 78% sensitivity and 80% specificity, confirming that the in vitro skin irritancy of the LabCyte EPI-MODEL correlates highly with in vivo skin irritation. These results suggest that LabCyte EPI-MODEL is applicable to the skin irritation testing protocol set out in the ECVAM Performance Standards.

Species Extrapolation of Life-Stage Physiologically-Based Pharmacokinetic (PBPK) Models to Investigate the Developmental Toxicology of Ethanol Using In vitro to In vivo (IVIVE) Methods

EPA Science Inventory

To provide useful alternatives to in vivo animal studies, in vitro assays for dose-response assessments of xenobiotic chemicals must use concentrations in media and target tissues that are within biologically-plausible limits. Determining these concentrations is a complex matter,...

Detection of ingested nitromethane and reliable creatinine assessment using multiple common analytical methods.

PubMed

Murphy, Christine M; Devlin, John J; Beuhler, Michael C; Cheifetz, Paul; Maynard, Susan; Schwartz, Michael D; Kacinko, Sherri

2018-04-01

Nitromethane, found in fuels used for short distance racing, model cars, and model airplanes, produces a falsely elevated serum creatinine with standard creatinine analysis via the Jaffé method. Erroneous creatinine elevation often triggers extensive testing, leads to inaccurate diagnoses, and delayed or inappropriate medical interventions. Multiple reports in the literature identify "enzymatic assays" as an alternative method to detect the true value of creatinine, but this ambiguity does not help providers translate what type of enzymatic assay testing can be done in real time to determine if there is indeed false elevation. We report seven cases of ingested nitromethane where creatinine was determined via Beckman Coulter ® analyser using the Jaffé method, Vitros ® analyser, or i-Stat ® point-of-care testing. Nitromethane was detected and semi-quantified using a common clinical toxic alcohol analysis method, and quantified by headspace-gas chromatography-mass spectrometry. When creatinine was determined using i-Stat ® point-of-care testing or a Vitros ® analyser, levels were within the normal range. Comparatively, all initial creatinine levels obtained via the Jaffé method were elevated. Nitromethane concentrations ranged from 42 to 310 μg/mL. These cases demonstrate reliable assessment of creatinine through other enzymatic methods using a Vitros ® analyser or i-STAT ® . Additionally, nitromethane is detectable and quantifiable using routine alcohols gas chromatography analysis and by headspace-gas chromatography-mass spectrometry.

Advantages and disadvantages of the animal models v. in vitro studies in iron metabolism: a review.

PubMed

García, Y; Díaz-Castro, J

2013-10-01

Iron deficiency is the most common nutritional deficiency in the world. Special molecules have evolved for iron acquisition, transport and storage in soluble, nontoxic forms. Studies about the effects of iron on health are focused on iron metabolism or nutrition to prevent or treat iron deficiency and anemia. These studies are focused in two main aspects: (1) basic studies to elucidate iron metabolism and (2) nutritional studies to evaluate the efficacy of iron supplementation to prevent or treat iron deficiency and anemia. This paper reviews the advantages and disadvantages of the experimental models commonly used as well as the methods that are more used in studies related to iron. In vitro studies have used different parts of the gut. In vivo studies are done in humans and animals such as mice, rats, pigs and monkeys. Iron metabolism is a complex process that includes interactions at the systemic level. In vitro studies, despite physiological differences to humans, are useful to increase knowledge related to this essential micronutrient. Isotopic techniques are the most recommended in studies related to iron, but their high cost and required logistic, making them difficult to use. The depletion-repletion of hemoglobin is a method commonly used in animal studies. Three depletion-repletion techniques are mostly used: hemoglobin regeneration efficiency, relative biological values (RBV) and metabolic balance, which are official methods of the association of official analytical chemists. These techniques are well-validated to be used as studies related to iron and their results can be extrapolated to humans. Knowledge about the main advantages and disadvantages of the in vitro and animal models, and methods used in these studies, could increase confidence of researchers in the experimental results with less costs.

Development of a solvent-free analytical method for paracetamol quantitative determination in Blood Brain Barrier in vitro model.

PubMed

Langlois, Marie-Hélène; Vekris, Antonios; Bousses, Christine; Mordelet, Elodie; Buhannic, Nathalie; Séguard, Céline; Couraud, Pierre-Olivier; Weksler, Babette B; Petry, Klaus G; Gaudin, Karen

2015-04-15

A Reversed Phase-High Performance Liquid Chromatography/Diode Array Detection method was developed and validated for paracetamol quantification in cell culture fluid from an in vitro Blood Brain Barrier model. The chromatographic method and sample preparation were developed using only aqueous solvents. The column was a XTerra RP18 150 × 4.6mm, 3.5 μm with a guard column XTerra RP18 20 × 4.6 mm, 3.5 μm at 35 °C and the mobile phase was composed by 100% formate buffer 20 mM at pH 4 and flow rate was set at 1 mL/min. The detection was at 242 nm. The sample was injected at 10 μL. Validation was performed using the accuracy profile approach. The analytical procedure was validated with the acceptance limits at ± 10% over a range of concentration from 1 to 58 mg L(-1). The procedure was then used in routine to determine paracetamol concentration in a brain blood barrier in vitro model. Application of the Unither paracetamol formulation in Blood Brain Barrier model allowed the determination and comparison of the transcellular passage of paracetamol at 37 °C and 4 °C, that excludes paracellular or non specific leakage. Copyright © 2015 Elsevier B.V. All rights reserved.

Arterial waveguide model for shear wave elastography: implementation and in vitro validation

NASA Astrophysics Data System (ADS)

Vaziri Astaneh, Ali; Urban, Matthew W.; Aquino, Wilkins; Greenleaf, James F.; Guddati, Murthy N.

2017-07-01

Arterial stiffness is found to be an early indicator of many cardiovascular diseases. Among various techniques, shear wave elastography has emerged as a promising tool for estimating local arterial stiffness through the observed dispersion of guided waves. In this paper, we develop efficient models for the computational simulation of guided wave dispersion in arterial walls. The models are capable of considering fluid-loaded tubes, immersed in fluid or embedded in a solid, which are encountered in in vitro/ex vivo, and in vivo experiments. The proposed methods are based on judiciously combining Fourier transformation and finite element discretization, leading to a significant reduction in computational cost while fully capturing complex 3D wave propagation. The developed methods are implemented in open-source code, and verified by comparing them with significantly more expensive, fully 3D finite element models. We also validate the models using the shear wave elastography of tissue-mimicking phantoms. The computational efficiency of the developed methods indicates the possibility of being able to estimate arterial stiffness in real time, which would be beneficial in clinical settings.

In-vitro evidence for efficacy of antimicrobial mouthrinses

PubMed Central

Pan, Pauline C.; Harper, Scott; Ricci-Nittel, Danette; Lux, Renate; Shi, Wenyuan

2010-01-01

SUMMARY Objectives The objective of this study was to compare the antimicrobial activity of commercially available antiseptic mouthrinses against saliva-derived plaque biofilms in static and flow-through biofilm systems in vitro. Methods Nine mouthrinses were tested in a recirculating flow-through biofilm model (RFTB) with viability assessment by ATP bioluminescence. In addition, five mouthrinses were evaluated in a batch chamber slide biofilm (BCSB) model, using live- dead staining and confocal laser scanning microscopy. Results In the RFTB model, essential oil (EO) and chlorhexidine (CHX)-containing rinses showed equivalent antimicrobial activity and were more effective than a range of cetyl pyridinium chloride (CPC1) formulations. In the BCSB model, twice-daily mouthrinse exposure demonstrated that the EO rinse was significantly more effective than rinses containing amine and stannous fluorides, a combination of CPC/CHX and CPC2. EO showed biofilm kill comparable to the CHX rinse. Conclusions The present studies have shown that mouthrinses vary significantly in their capability to kill plaque biofilm bacteria in BCSB and RFTB models. The EO mouthrinse demonstrated superior antiplaque biofilm activity to AFSF, CPC/CHX, and CPC rinses and comparable activity to CHX. The methods tested may be of value for the in-vitro screening of antiseptic rinses with different modes of antimicrobial action. PMID:20621239

AlgiMatrix™-Based 3D Cell Culture System as an In Vitro Tumor Model: An Important Tool in Cancer Research.

PubMed

Godugu, Chandraiah; Singh, Mandip

2016-01-01

Routinely used two-dimensional cell culture-based models often fail while translating the observations into in vivo models. This setback is more common in cancer research, due to several reasons. The extracellular matrix and cell-to-cell interactions are not present in two-dimensional (2D) cell culture models. Diffusion of drug molecules into cancer cells is hindered by barriers of extracellular components in in vivo conditions, these barriers are absent in 2D cell culture models. To better mimic or simulate the in vivo conditions present in tumors, the current study used the alginate based three-dimensional cell culture (AlgiMatrix™) model, which resembles close to the in vivo tumor models. The current study explains the detailed protocols involved in AlgiMatrix™ based in vitro non-small-cell lung cancer (NSCLC) models. The suitability of this model was studied by evaluating, cytotoxicity, apoptosis, and penetration of nanoparticles into the in vitro tumor spheroids. This study also demonstrated the effect of EphA2 receptor targeted docetaxel-loaded nanoparticles on MDA-MB-468 TNBC cell lines. The methods section is subdivided into three subsections such as (1) preparation of AlgiMatrix™-based 3D in vitro tumor models and cytotoxicity assays, (2) free drug and nanoparticle uptake into spheroid studies, and (3) western blot, IHC, and RT-PCR studies.

Engineering an in vitro air-blood barrier by 3D bioprinting

PubMed Central

Horváth, Lenke; Umehara, Yuki; Jud, Corinne; Blank, Fabian; Petri-Fink, Alke; Rothen-Rutishauser, Barbara

2015-01-01

Intensive efforts in recent years to develop and commercialize in vitro alternatives in the field of risk assessment have yielded new promising two- and three dimensional (3D) cell culture models. Nevertheless, a realistic 3D in vitro alveolar model is not available yet. Here we report on the biofabrication of the human air-blood tissue barrier analogue composed of an endothelial cell, basement membrane and epithelial cell layer by using a bioprinting technology. In contrary to the manual method, we demonstrate that this technique enables automatized and reproducible creation of thinner and more homogeneous cell layers, which is required for an optimal air-blood tissue barrier. This bioprinting platform will offer an excellent tool to engineer an advanced 3D lung model for high-throughput screening for safety assessment and drug efficacy testing. PMID:25609567

Application of a Physiologically Based Pharmacokinetic Model to Assess Propofol Hepatic and Renal Glucuronidation in Isolation: Utility of In Vitro and In Vivo Data

PubMed Central

Gill, Katherine L.; Gertz, Michael; Houston, J. Brian

2013-01-01

A physiologically based pharmacokinetic (PBPK) modeling approach was used to assess the prediction accuracy of propofol hepatic and extrahepatic metabolic clearance and to address previously reported underprediction of in vivo clearance based on static in vitro–in vivo extrapolation methods. The predictive capacity of propofol intrinsic clearance data (CLint) obtained in human hepatocytes and liver and kidney microsomes was assessed using the PBPK model developed in MATLAB software. Microsomal data obtained by both substrate depletion and metabolite formation methods and in the presence of 2% bovine serum albumin were considered in the analysis. Incorporation of hepatic and renal in vitro metabolic clearance in the PBPK model resulted in underprediction of propofol clearance regardless of the source of in vitro data; the predicted value did not exceed 35% of the observed clearance. Subsequently, propofol clinical data from three dose levels in intact patients and anhepatic subjects were used for the optimization of hepatic and renal CLint in a simultaneous fitting routine. Optimization process highlighted that renal glucuronidation clearance was underpredicted to a greater extent than liver clearance, requiring empirical scaling factors of 17 and 9, respectively. The use of optimized clearance parameters predicted hepatic and renal extraction ratios within 20% of the observed values, reported in an additional independent clinical study. This study highlights the complexity involved in assessing the contribution of extrahepatic clearance mechanisms and illustrates the application of PBPK modeling, in conjunction with clinical data, to assess prediction of clearance from in vitro data for each tissue individually. PMID:23303442

Cheminformatics Analysis of EPA ToxCast Chemical Libraries to Identify Domains of Applicability for Predictive Toxicity Models and Prioritize Compounds for Toxicity Testing

EPA Science Inventory

An important goal of toxicology research is the development of robust methods that use in vitro and chemical structure information to predict in vivo toxicity endpoints. The US EPA ToxCast program is addressing this goal using ~600 in vitro assays to create bioactivity profiles o...

Seed coat removal improves Fe bioavailability in cooked lentils: studies using an in vitro digestion/Caco-2 cell culture model

USDA-ARS?s Scientific Manuscript database

This study examined the range of Fe concentration and relative Fe bioavailability of 24 varieties of cooked lentils, as well as the impact of seed coat removal on lentil Fe nutritional quality. Relative Fe bioavailability was assessed by the in vitro/Caco-2 cell culture method. While Fe concentrat...

Factors Affecting the Longevity and Strength in an In Vitro Model of the Bone–Ligament Interface

PubMed Central

Paxton, Jennifer Z.; Donnelly, Kenneth; Keatch, Robert P.; Grover, Liam M.

2010-01-01

The interfaces between musculoskeletal tissues with contrasting moduli are morphologically and biochemically adapted to allow the transmission of force with minimal injury. Current methods of tissue engineering ligaments and tendons do not include the interface and this may limit the future clinical success of engineered musculoskeletal tissues. This study aimed to use solid brushite cement anchors to engineer intact ligaments from bone-to-bone, creating a functional musculoskeletal interface in vitro. We show here that modifying anchor shape and cement composition can alter both the longevity and the strength of an in vitro model of the bone–ligament interface: with values reaching 23 days and 21.6 kPa, respectively. These results validate the use of brushite bone cement to engineer the bone–ligament interface in vitro and raise the potential for future use in ligament replacement surgery. PMID:20431953

In vitro test systems supporting the development of improved pest control methods: a case study with chemical mixtures and bivalve biofoulers.

PubMed

Silva, Carlos; Nunes, Bruno; Nogueira, António Ja; Gonçalves, Fernando; Pereira, Joana L

2016-11-01

Using the bivalve macrofouler Corbicula fluminea, the suitability of in vitro testing as a stepping stone towards the improvement of control methods based on chemical mixtures was addressed in this study. In vitro cholinesterase (ChE) activity inhibition following single exposure of C. fluminea tissue to four model chemicals (the organophosphates dimethoate and dichlorvos, copper and sodium dodecyl phosphate [SDS]) was first assessed. Consequently, mixtures of dimethoate with copper and dichlorvos with SDS were tested and modelled; mixtures with ChE revealed synergistic interactions for both chemical pairs. These synergic combinations were subsequently validated in vivo and the increased control potential of these selected combinations was verified, with gains of up to 50% in C. fluminea mortality relative to corresponding single chemical treatments. Such consistency supports the suitability of using time- and cost-effective surrogate testing platforms to assist the development of biofouling control strategies incorporating mixtures.

In vitro culture and characterization of human lung cancer circulating tumor cells isolated by size exclusion from an orthotopic nude-mouse model expressing fluorescent protein.

PubMed

Kolostova, Katarina; Zhang, Yong; Hoffman, Robert M; Bobek, Vladimir

2014-09-01

In the present study, we demonstrate an animal model and recently introduced size-based exclusion method for circulating tumor cells (CTCs) isolation. The methodology enables subsequent in vitro CTC-culture and characterization. Human lung cancer cell line H460, expressing red fluorescent protein (H460-RFP), was orthotopically implanted in nude mice. CTCs were isolated by a size-based filtration method and successfully cultured in vitro on the separating membrane (MetaCell®), analyzed by means of time-lapse imaging. The cultured CTCs were heterogeneous in size and morphology even though they originated from a single tumor. The outer CTC-membranes were blebbing in general. Abnormal mitosis resulting in three daughter cells was frequently observed. The expression of RFP ensured that the CTCs originated from lung tumor. These readily isolatable, identifiable and cultivable CTCs can be used to characterize individual patient cancers and for screening of more effective treatment.

A three dimensional in vitro glial scar model to investigate the local strain effects from micromotion around neural implants.

PubMed

Spencer, Kevin C; Sy, Jay C; Falcón-Banchs, Roberto; Cima, Michael J

2017-02-28

Glial scar formation remains a significant barrier to the long term success of neural probes. Micromotion coupled with mechanical mismatch between the probe and tissue is believed to be a key driver of the inflammatory response. In vitro glial scar models present an intermediate step prior to conventional in vivo histology experiments as they enable cell-device interactions to be tested on a shorter timescale, with the ability to conduct broader biochemical assays. No established in vitro models have incorporated methods to assess device performance with respect to mechanical factors. In this study, we describe an in vitro glial scar model that combines high-precision linear actuators to simulate axial micromotion around neural implants with a 3D primary neural cell culture in a collagen gel. Strain field measurements were conducted to visualize the local displacement within the gel in response to micromotion. Primary brain cell cultures were found to be mechanically responsive to micromotion after one week in culture. Astrocytes, as determined by immunohistochemical staining, were found to have significantly increased in cell areas and perimeters in response to micromotion compared to static control wells. These results demonstrate the importance of micromotion when considering the chronic response to neural implants. Going forward, this model provides advantages over existing in vitro models as it will enable critical mechanical design factors of neural implants to be evaluated prior to in vivo testing.

Developments in Methods for Measuring the Intestinal Absorption of Nanoparticle-Bound Drugs

PubMed Central

Liu, Wei; Pan, Hao; Zhang, Caiyun; Zhao, Liling; Zhao, Ruixia; Zhu, Yongtao; Pan, Weisan

2016-01-01

With the rapid development of nanotechnology, novel drug delivery systems comprising orally administered nanoparticles (NPs) have been paid increasing attention in recent years. The bioavailability of orally administered drugs has significant influence on drug efficacy and therapeutic dosage, and it is therefore imperative that the intestinal absorption of oral NPs be investigated. This review examines the various literature on the oral absorption of polymeric NPs, and provides an overview of the intestinal absorption models that have been developed for the study of oral nanoparticles. Three major categories of models including a total of eight measurement methods are described in detail (in vitro: dialysis bag, rat gut sac, Ussing chamber, cell culture model; in situ: intestinal perfusion, intestinal loops, intestinal vascular cannulation; in vivo: the blood/urine drug concentration method), and the advantages and disadvantages of each method are contrasted and elucidated. In general, in vitro and in situ methods are relatively convenient but lack accuracy, while the in vivo method is troublesome but can provide a true reflection of drug absorption in vivo. This review summarizes the development of intestinal absorption experiments in recent years and provides a reference for the systematic study of the intestinal absorption of nanoparticle-bound drugs. PMID:27455239

Hanging drop: an in vitro air toxic exposure model using human lung cells in 2D and 3D structures.

PubMed

Liu, Faye F; Peng, Cheng; Escher, Beate I; Fantino, Emmanuelle; Giles, Cindy; Were, Stephen; Duffy, Lesley; Ng, Jack C

2013-10-15

Using benzene as a candidate air toxicant and A549 cells as an in vitro cell model, we have developed and validated a hanging drop (HD) air exposure system that mimics an air liquid interface exposure to the lung for periods of 1h to over 20 days. Dose response curves were highly reproducible for 2D cultures but more variable for 3D cultures. By comparing the HD exposure method with other classically used air exposure systems, we found that the HD exposure method is more sensitive, more reliable and cheaper to run than medium diffusion methods and the CULTEX(®) system. The concentration causing 50% of reduction of cell viability (EC50) for benzene, toluene, p-xylene, m-xylene and o-xylene to A549 cells for 1h exposure in the HD system were similar to previous in vitro static air exposure. Not only cell viability could be assessed but also sub lethal biological endpoints such as DNA damage and interleukin expressions. An advantage of the HD exposure system is that bioavailability and cell concentrations can be derived from published physicochemical properties using a four compartment mass balance model. The modelled cellular effect concentrations EC50cell for 1h exposure were very similar for benzene, toluene and three xylenes and ranged from 5 to 15 mmol/kgdry weight, which corresponds to the intracellular concentration of narcotic chemicals in many aquatic species, confirming the high sensitivity of this exposure method. Copyright © 2013 Elsevier B.V. All rights reserved.

Pediatric in vitro and in silico models of deposition via oral and nasal inhalation.

PubMed

Carrigy, Nicholas B; Ruzycki, Conor A; Golshahi, Laleh; Finlay, Warren H

2014-06-01

Respiratory tract deposition models provide a useful method for optimizing the design and administration of inhaled pharmaceutical aerosols, and can be useful for estimating exposure risks to inhaled particulate matter. As aerosol must first pass through the extrathoracic region prior to reaching the lungs, deposition in this region plays an important role in both cases. Compared to adults, much less extrathoracic deposition data are available with pediatric subjects. Recently, progress in magnetic resonance imaging and computed tomography scans to develop pediatric extrathoracic airway replicas has facilitated addressing this issue. Indeed, the use of realistic replicas for benchtop inhaler testing is now relatively common during the development and in vitro evaluation of pediatric respiratory drug delivery devices. Recently, in vitro empirical modeling studies using a moderate number of these realistic replicas have related airway geometry, particle size, fluid properties, and flow rate to extrathoracic deposition. Idealized geometries provide a standardized platform for inhaler testing and exposure risk assessment and have been designed to mimic average in vitro deposition in infants and children by replicating representative average geometrical dimensions. In silico mathematical models have used morphometric data and aerosol physics to illustrate the relative importance of different deposition mechanisms on respiratory tract deposition. Computational fluid dynamics simulations allow for the quantification of local deposition patterns and an in-depth examination of aerosol behavior in the respiratory tract. Recent studies have used both in vitro and in silico deposition measurements in realistic pediatric airway geometries to some success. This article reviews the current understanding of pediatric in vitro and in silico deposition modeling via oral and nasal inhalation.

[In vitro metabolism of fenbendazole prodrug].

PubMed

Wen, Ai-Dan; Duan, Li-Ping; Liu, Cong-Shan; Tao, Yi; Xue, Jian; Wu, Ning-Bo; Jiang, Bin; Zhang, Hao-Bing

2013-02-01

Synthesized fenbendazole prodrug N-methoxycarbonyl-N'-(2-nitro-4-phenylthiophenyl) thiourea (MPT) was analyzed in vitro in artificial gastric juice, intestinal juice and mouse liver homogenate model by using HPLC method, and metabolic curve was then generated. MPT was tested against Echinococcus granulosus protoscolices in vitro. The result showed that MPT could be metabolized in the three biological media, and to the active compound fenbendazole in liver homogenate, with a metabolic rate of 7.92%. Besides, the prodrug showed a weak activity against E. granulosus protoscolices with a mortality of 45.9%.

Multiscale modelling approaches for assessing cosmetic ingredients safety.

PubMed

Bois, Frédéric Y; Ochoa, Juan G Diaz; Gajewska, Monika; Kovarich, Simona; Mauch, Klaus; Paini, Alicia; Péry, Alexandre; Benito, Jose Vicente Sala; Teng, Sophie; Worth, Andrew

2017-12-01

The European Union's ban on animal testing for cosmetic ingredients and products has generated a strong momentum for the development of in silico and in vitro alternative methods. One of the focus of the COSMOS project was ab initio prediction of kinetics and toxic effects through multiscale pharmacokinetic modeling and in vitro data integration. In our experience, mathematical or computer modeling and in vitro experiments are complementary. We present here a summary of the main models and results obtained within the framework of the project on these topics. A first section presents our work at the organelle and cellular level. We then go toward modeling cell levels effects (monitored continuously), multiscale physiologically based pharmacokinetic and effect models, and route to route extrapolation. We follow with a short presentation of the automated KNIME workflows developed for dissemination and easy use of the models. We end with a discussion of two challenges to the field: our limited ability to deal with massive data and complex computations. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Non-animal approaches for toxicokinetics in risk evaluations of food chemicals.

PubMed

Punt, Ans; Peijnenburg, Ad A C M; Hoogenboom, Ron L A P; Bouwmeester, Hans

2017-01-01

The objective of the present work was to review the availability and predictive value of non-animal toxicokinetic approaches and to evaluate their current use in European risk evaluations of food contaminants, additives and food contact materials, as well as pesticides and medicines. Results revealed little use of quantitative animal or human kinetic data in risk evaluations of food chemicals, compared with pesticides and medicines. Risk evaluations of medicines provided sufficient in vivo kinetic data from different species to evaluate the predictive value of animal kinetic data for humans. These data showed a relatively poor correlation between the in vivo bioavailability in rats and dogs versus that in humans. In contrast, in vitro (human) kinetic data have been demonstrated to provide adequate predictions of the fate of compounds in humans, using appropriate in vitro-in vivo scalers and by integration of in vitro kinetic data with in silico kinetic modelling. Even though in vitro kinetic data were found to be occasionally included within risk evaluations of food chemicals, particularly results from Caco-2 absorption experiments and in vitro data on gut-microbial conversions, only minor use of in vitro methods for metabolism and quantitative in vitro-in vivo extrapolation methods was identified. Yet, such quantitative predictions are essential in the development of alternatives to animal testing as well as to increase human relevance of toxicological risk evaluations. Future research should aim at further improving and validating quantitative alternative methods for kinetics, thereby increasing regulatory acceptance of non-animal kinetic data.

Establishment of a new in vitro test method for evaluation of eye irritancy using a reconstructed human corneal epithelial model, LabCyte CORNEA-MODEL.

PubMed

Katoh, Masakazu; Hamajima, Fumiyasu; Ogasawara, Takahiro; Hata, Ken-ichiro

2013-12-01

Finding in vitro eye irritation testing alternatives to animal testing such as the Draize eye test, which uses rabbits, is essential from the standpoint of animal welfare. It has been developed a reconstructed human corneal epithelial model, the LabCyte CORNEA-MODEL, which has a representative corneal epithelium-like structure. Protocol optimization (pre-validation study) was examined in order to establish a new alternative method for eye irritancy evaluation with this model. From the results of the optimization experiments, the application periods for chemicals were set at 1min for liquid chemicals or 24h for solid chemicals, and the post-exposure incubation periods were set at 24h for liquids or zero for solids. If the viability was less than 50%, the chemical was judged to be an eye irritant. Sixty-one chemicals were applied in the optimized protocol using the LabCyte CORNEA-MODEL and these results were evaluated in correlation with in vivo results. The predictions of the optimized LabCyte CORNEA-MODEL eye irritation test methods were highly correlated with in vivo eye irritation (sensitivity 100%, specificity 80.0%, and accuracy 91.8%). These results suggest that the LabCyte CORNEA-MODEL eye irritation test could be useful as an alternative method to the Draize eye test. Copyright © 2013 Elsevier Ltd. All rights reserved.

Extended release dosage form of glipizide: development and validation of a level A in vitro-in vivo correlation.

PubMed

Ghosh, Animesh; Bhaumik, Uttam Kumar; Bose, Anirbandeep; Mandal, Uttam; Gowda, Veeran; Chatterjee, Bappaditya; Chakrabarty, Uday Sankar; Pal, Tapan Kumar

2008-10-01

Defining a quantitative and reliable relationship between in vitro drug release and in vivo absorption is highly desired for rational development, optimization, and evaluation of controlled-release dosage forms and manufacturing process. During the development of once daily extended-release (ER) tablet of glipizide, a predictive in vitro drug release method was designed and statistically evaluated using three formulations with varying release rates. In order to establish internally and externally validated level A in vitro-in vivo correlation (IVIVC), a total of three different ER formulations of glipizide were used to evaluate a linear IVIVC model based on the in vitro test method. For internal validation, a single-dose four-way cross over study (n=6) was performed using fast-, moderate-, and slow-releasing ER formulations and an immediate-release (IR) of glipizide as reference. In vitro release rate data were obtained for each formulation using the United States Pharmacopeia (USP) apparatus II, paddle stirrer at 50 and 100 rev. min(-1) in 0.1 M hydrochloric acid (HCl) and pH 6.8 phosphate buffer. The f(2) metric (similarity factor) was used to analyze the dissolution data. The formulations were compared using area under the plasma concentration-time curve, AUC(0-infinity), time to reach peak plasma concentration, T(max), and peak plasma concentration, C(max), while correlation was determined between in vitro release and in vivo absorption. A linear correlation model was developed using percent absorbed data versus percent dissolved from the three formulations. Predicted glipizide concentrations were obtained by convolution of the in vivo absorption rates. Prediction errors were estimated for C(max) and AUC(0-infinity) to determine the validity of the correlation. Apparatus II, pH 6.8 at 100 rev. min(-1) was found to be the most discriminating dissolution method. Linear regression analysis of the mean percentage of dose absorbed versus the mean percentage of in vitro release resulted in a significant correlation (r(2)>or=0.9) for the three formulations.

Ex vitro composite plants: an inexpensive, rapid method for root biology.

PubMed

Collier, Ray; Fuchs, Beth; Walter, Nathalie; Kevin Lutke, William; Taylor, Christopher G

2005-08-01

Plant transformation technology is frequently the rate-limiting step in gene function analysis in non-model plants. An important tool for root biologists is the Agrobacterium rhizogenes-derived composite plant, which has made possible genetic analyses in a wide variety of transformation recalcitrant dicotyledonous plants. The novel, rapid and inexpensive ex vitro method for producing composite plants described in this report represents a significant advance over existing composite plant induction protocols, which rely on expensive and time-consuming in vitro conditions. The utility of the new system is validated by expression and RNAi silencing of GFP in transgenic roots of composite plants, and is bolstered further by experimental disruption, via RNAi silencing, of endogenous plant resistance to the plant parasitic nematode Meloidogyne incognita in transgenic roots of Lycopersicon esculentum cv. Motelle composite plants. Critical parameters of the method are described and discussed herein.

Gold nanoparticles synthesis and biological activity estimation in vitro and in vivo.

PubMed

Rieznichenko, L S; Dybkova, S M; Gruzina, T G; Ulberg, Z R; Todor, I N; Lukyanova, N Yu; Shpyleva, S I; Chekhun, V F

2012-01-01

The aim of the work was the synthesis of gold nanoparticles (GNP) of different sizes and the estimation of their biological activity in vitro and in vivo. Water dispersions of gold nanoparticles of different sizes have been synthesized by Davis method and characterized by laser-correlation spectroscopy and transmission electron microscopy methods. The GNP interaction with tumor cells has been visualized by confocal microscopy method. The enzyme activity was determined by standard biochemical methods. GNP distribution and content in organs and tissues have been determined via atomic-absorption spectrometry method; genotoxic influence has been estimated by "Comet-assay" method. The GNP size-dependent accumulation in cultured U937 tumor cells and their ability to modulate U937 cell membrane Na(+),K(+)-АТР-ase activity value has been revealed in vitro. Using in vivo model of Guerin carcinoma it has been shown that GNP possess high affinity to tumor cells. Our results indicate the perspectives of use of the synthesized GNP water dispersions for cancer diagnostics and treatment. It's necessary to take into account a size-dependent biosafety level of nanoparticles.

Workgroup Report: Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity into International Hazard and Risk Assessment Strategies

PubMed Central

Coecke, Sandra; Goldberg, Alan M; Allen, Sandra; Buzanska, Leonora; Calamandrei, Gemma; Crofton, Kevin; Hareng, Lars; Hartung, Thomas; Knaut, Holger; Honegger, Paul; Jacobs, Miriam; Lein, Pamela; Li, Abby; Mundy, William; Owen, David; Schneider, Steffen; Silbergeld, Ellen; Reum, Torsten; Trnovec, Tomas; Monnet-Tschudi, Florianne; Bal-Price, Anna

2007-01-01

This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19–21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work. PMID:17589601

A level A in vitro/in vivo correlation in fasted and fed states using different methods: applied to solid immediate release oral dosage form.

PubMed

Souliman, Sabah; Blanquet, Stéphanie; Beyssac, Eric; Cardot, Jean-Michel

2006-01-01

The first purpose of this study was to simulate the impact of food intake on drug release and absorption in vivo using a novel in vitro system which mimics the gastro-intestinal (GI) tract in man. The drug studied was acetaminophen in the form of immediate release (IR) tablets. The second purpose was to establish a level A in vitro/in vivo correlation that could predict the bioavailability of a drug instead of using difficult, time-consuming and expensive in vivo bioequivalence studies. The artificial digestive system was used to estimate the availability of acetaminophen IR tablets for absorption in fasted and fed states. The same study was performed in vivo under similar conditions. A comparison study was carried out between the classical and the novel methods to estimate the efficacy of the new in vitro system to simulate the influence of food on drug release and absorption in vivo. A level A in vitro/in vivo correlation was established with a correlation coefficient of 0.9128 and 0.9984 in the fasted and fed states, respectively. Compared to USP II method, the novel in vitro model demonstrated a high level of efficacy in mimicking the behaviour of acetaminophen IR tablets in vivo in fasted and fed states.

Development of in vitro-in vivo correlation of parenteral naltrexone loaded polymeric microspheres.

PubMed

Andhariya, Janki V; Shen, Jie; Choi, Stephanie; Wang, Yan; Zou, Yuan; Burgess, Diane J

2017-06-10

Establishment of in vitro-in vivo correlations (IVIVCs) for parenteral polymeric microspheres has been very challenging, due to their complex multiphase release characteristics (which is affected by the nature of the drug) as well as the lack of compendial in vitro release testing methods. Previously, a Level A correlation has been established and validated for polymeric microspheres containing risperidone (a practically water insoluble small molecule drug). The objectives of the present study were: 1) to investigate whether a Level A IVIVC can be established for polymeric microspheres containing another small molecule drug with different solubility profiles compared to risperidone; and 2) to determine whether release characteristic differences (bi-phasic vs tri-phasic) between microspheres can affect the development and predictability of IVIVCs. Naltrexone was chosen as the model drug. Three compositionally equivalent formulations of naltrexone microspheres with different release characteristics were prepared using different manufacturing processes. The critical physicochemical properties (such as drug loading, particle size, porosity, and morphology) as well as the in vitro release characteristics of the prepared naltrexone microspheres and the reference-listed drug (Vivitrol®) were determined. The pharmacokinetics of the naltrexone microspheres were investigated using a rabbit model. The obtained pharmacokinetic profiles were deconvoluted using the Loo-Riegelman method, and compared with the in vitro release profiles of the naltrexone microspheres obtained using USP apparatus 4. Level A IVIVCs were established and validated for predictability. The results demonstrated that the developed USP 4 method was capable of detecting manufacturing process related performance changes, and most importantly, predicting the in vivo performance of naltrexone microspheres in the investigated animal model. A critical difference between naltrexone and risperidone loaded microspheres is their respective bi-phasic and tri-phasic release profiles with varying burst release and lag phase. These variations in release profiles affect the development of IVIVCs. Nevertheless, IVIVCs have been established and validated for polymeric microspheres with different release characteristics. Copyright © 2017. Published by Elsevier B.V.

Permeability of PEGylated immunoarsonoliposomes through in vitro blood brain barrier-medulloblastoma co-culture models for brain tumor therapy.

PubMed

Al-Shehri, Abdulghani; Favretto, Marco E; Ioannou, Panayiotis V; Romero, Ignacio A; Couraud, Pierre-Olivier; Weksler, Babette Barbash; Parker, Terry L; Kallinteri, Paraskevi

2015-03-01

Owing to restricted access of pharmacological agents into the brain due to blood brain barrier (BBB) there is a need: 1. to develop a more representative 3-D-co-culture model of tumor-BBB interaction to investigate drug and nanoparticle transport into the brain for diagnostic and therapeutic evaluation. 2. to address the lack of new alternative methods to animal testing according to replacement-reduction-refinement principles. In this work, in vitro BBB-medulloblastoma 3-D-co-culture models were established using immortalized human primary brain endothelial cells (hCMEC/D3). hCMEC/D3 cells were cultured in presence and in absence of two human medulloblastoma cell lines on Transwell membranes. In vitro models were characterized for BBB formation, zonula occludens-1 expression and permeability to dextran. Transferrin receptors (Tfr) expressed on hCMEC/D3 were exploited to facilitate arsonoliposome (ARL) permeability through the BBB to the tumor by covalently attaching an antibody specific to human Tfr. The effect of anticancer ARLs on hCMEC/D3 was assessed. In vitro BBB and BBB-tumor co-culture models were established successfully. BBB permeability was affected by the presence of tumor aggregates as suggested by increased permeability of ARLs. There was a 6-fold and 8-fold increase in anti-Tfr-ARL uptake into VC312R and BBB-DAOY co-culture models, respectively, compared to plain ARLs. The three-dimensional models might be appropriate models to study the transport of various drugs and nanocarriers (liposomes and immunoarsonoliposomes) through the healthy and diseased BBB. The immunoarsonoliposomes can be potentially used as anticancer agents due to good tolerance of the in vitro BBB model to their toxic effect.

In Vitro Analysis of the Role of Schwann Cells on Axonal Degeneration and Regeneration Using Sensory Neurons from Dorsal Root Ganglia.

PubMed

López-Leal, Rodrigo; Diaz, Paula; Court, Felipe A

2018-01-01

Sensory neurons from dorsal root ganglion efficiently regenerate after peripheral nerve injuries. These neurons are widely used as a model system to study degenerative mechanisms of the soma and axons, as well as regenerative axonal growth in the peripheral nervous system. This chapter describes techniques associated to the study of axonal degeneration and regeneration using explant cultures of dorsal root ganglion sensory neurons in vitro in the presence or absence of Schwann cells. Schwann cells are extremely important due to their involvement in tissue clearance during axonal degeneration as well as their known pro-regenerative effect during regeneration in the peripheral nervous system. We describe methods to induce and study axonal degeneration triggered by axotomy (mechanical separation of the axon from its soma) and treatment with vinblastine (which blocks axonal transport), which constitute clinically relevant mechanical and toxic models of axonal degeneration. In addition, we describe three different methods to evaluate axonal regeneration using quantitative methods. These protocols constitute a valuable tool to analyze in vitro mechanisms associated to axonal degeneration and regeneration of sensory neurons and the role of Schwann cells in these processes.

Rationalizing the selection of oral lipid based drug delivery systems by an in vitro dynamic lipolysis model for improved oral bioavailability of poorly water soluble drugs.

PubMed

Dahan, Arik; Hoffman, Amnon

2008-07-02

As a consequence of modern drug discovery techniques, there has been a consistent increase in the number of new pharmacologically active lipophilic compounds that are poorly water soluble. A great challenge facing the pharmaceutical scientist is making these molecules into orally administered medications with sufficient bioavailability. One of the most popular approaches to improve the oral bioavailability of these molecules is the utilization of a lipid based drug delivery system. Unfortunately, current development strategies in the area of lipid based delivery systems are mostly empirical. Hence, there is a need for a simplified in vitro method to guide the selection of a suitable lipidic vehicle composition and to rationalize the delivery system design. To address this need, a dynamic in vitro lipolysis model, which provides a very good simulation of the in vivo lipid digestion process, has been developed over the past few years. This model has been extensively used for in vitro assessment of different lipid based delivery systems, leading to enhanced understanding of the suitability of different lipids and surfactants as a delivery system for a given poorly water soluble drug candidate. A key goal in the development of the dynamic in vitro lipolysis model has been correlating the in vitro data of various drug-lipidic delivery system combinations to the resultant in vivo drug profile. In this paper, we discuss and review the need for this model, its underlying theory, practice and limitations, and the available data accumulated in the literature. Overall, the dynamic in vitro lipolysis model seems to provide highly useful initial guidelines in the development process of oral lipid based drug delivery systems for poorly water soluble drugs, and it predicts phenomena that occur in the pre-enterocyte stages of the intestinal absorption cascade.

Machine Learning Model Analysis and Data Visualization with Small Molecules Tested in a Mouse Model of Mycobacterium tuberculosis Infection (2014–2015)

PubMed Central

2016-01-01

The renewed urgency to develop new treatments for Mycobacterium tuberculosis (Mtb) infection has resulted in large-scale phenotypic screening and thousands of new active compounds in vitro. The next challenge is to identify candidates to pursue in a mouse in vivo efficacy model as a step to predicting clinical efficacy. We previously analyzed over 70 years of this mouse in vivo efficacy data, which we used to generate and validate machine learning models. Curation of 60 additional small molecules with in vivo data published in 2014 and 2015 was undertaken to further test these models. This represents a much larger test set than for the previous models. Several computational approaches have now been applied to analyze these molecules and compare their molecular properties beyond those attempted previously. Our previous machine learning models have been updated, and a novel aspect has been added in the form of mouse liver microsomal half-life (MLM t1/2) and in vitro-based Mtb models incorporating cytotoxicity data that were used to predict in vivo activity for comparison. Our best Mtbin vivo models possess fivefold ROC values > 0.7, sensitivity > 80%, and concordance > 60%, while the best specificity value is >40%. Use of an MLM t1/2 Bayesian model affords comparable results for scoring the 60 compounds tested. Combining MLM stability and in vitroMtb models in a novel consensus workflow in the best cases has a positive predicted value (hit rate) > 77%. Our results indicate that Bayesian models constructed with literature in vivoMtb data generated by different laboratories in various mouse models can have predictive value and may be used alongside MLM t1/2 and in vitro-based Mtb models to assist in selecting antitubercular compounds with desirable in vivo efficacy. We demonstrate for the first time that consensus models of any kind can be used to predict in vivo activity for Mtb. In addition, we describe a new clustering method for data visualization and apply this to the in vivo training and test data, ultimately making the method accessible in a mobile app. PMID:27335215

Pharmacokinetic/pharmacodynamic modelling approaches in paediatric infectious diseases and immunology☆

PubMed Central

Barker, Charlotte I.S.; Germovsek, Eva; Hoare, Rollo L.; Lestner, Jodi M.; Lewis, Joanna; Standing, Joseph F.

2014-01-01

Pharmacokinetic/pharmacodynamic (PKPD) modelling is used to describe and quantify dose–concentration–effect relationships. Within paediatric studies in infectious diseases and immunology these methods are often applied to developing guidance on appropriate dosing. In this paper, an introduction to the field of PKPD modelling is given, followed by a review of the PKPD studies that have been undertaken in paediatric infectious diseases and immunology. The main focus is on identifying the methodological approaches used to define the PKPD relationship in these studies. The major findings were that most studies of infectious diseases have developed a PK model and then used simulations to define a dose recommendation based on a pre-defined PD target, which may have been defined in adults or in vitro. For immunological studies much of the modelling has focused on either PK or PD, and since multiple drugs are usually used, delineating the relative contributions of each is challenging. The use of dynamical modelling of in vitro antibacterial studies, and paediatric HIV mechanistic PD models linked with the PK of all drugs, are emerging methods that should enhance PKPD-based recommendations in the future. PMID:24440429

Videodensitometric Methods for Cardiac Output Measurements

NASA Astrophysics Data System (ADS)

Mischi, Massimo; Kalker, Ton; Korsten, Erik

2003-12-01

Cardiac output is often measured by indicator dilution techniques, usually based on dye or cold saline injections. Developments of more stable ultrasound contrast agents (UCA) are leading to new noninvasive indicator dilution methods. However, several problems concerning the interpretation of dilution curves as detected by ultrasound transducers have arisen. This paper presents a method for blood flow measurements based on UCA dilution. Dilution curves are determined by real-time densitometric analysis of the video output of an ultrasound scanner and are automatically fitted by the Local Density Random Walk model. A new fitting algorithm based on multiple linear regression is developed. Calibration, that is, the relation between videodensity and UCA concentration, is modelled by in vitro experimentation. The flow measurement system is validated by in vitro perfusion of SonoVue contrast agent. The results show an accurate dilution curve fit and flow estimation with determination coefficient larger than 0.95 and 0.99, respectively.

Analysis methods and recent advances in nonlinear pharmacokinetics from in vitro through in loci to in vivo.

PubMed

Yamaoka, Kiyoshi; Takakura, Yoshinobu

2004-12-01

An attempt has been made to review the nonlinearities in the disposition in vitro, in situ, in loci and in vivo mainly from a theoretical point of view. Parallel Michaelis-Menten and linear (first-order) eliminations are often observed in the cellular uptake, metabolism and efflux of drugs. The well-stirred and parallel-tube models are mainly adopted under steady-state conditions in perfusion experiments, whereas distribution, tank-in-series and dispersion models are often used under nonsteady-state conditions with a pulse input. The analysis of the nonlinear local disposition in loci is reviewed from two points of view, namely an indirect method involving physiologically based pharmacokinetics (PBPK) and a direct (two or three samplings) method using live animals. The nonlinear global pharmacokinetics in vivo is reviewed with regard to absorption, elimination (metabolism and excretion) and distribution.

Development of X-ray micro-focus computed tomography to image and quantify biofilms in central venous catheter models in vitro.

PubMed

Niehaus, Wilmari L; Howlin, Robert P; Johnston, David A; Bull, Daniel J; Jones, Gareth L; Calton, Elizabeth; Mavrogordato, Mark N; Clarke, Stuart C; Thurner, Philipp J; Faust, Saul N; Stoodley, Paul

2016-09-01

Bacterial infections of central venous catheters (CVCs) cause much morbidity and mortality, and are usually diagnosed by concordant culture of blood and catheter tip. However, studies suggest that culture often fails to detect biofilm bacteria. This study optimizes X-ray micro-focus computed tomography (X-ray µCT) for the quantification and determination of distribution and heterogeneity of biofilms in in vitro CVC model systems.Bacterial culture and scanning electron microscopy (SEM) were used to detect Staphylococcus epidermidis ATCC 35984 biofilms grown on catheters in vitro in both flow and static biofilm models. Alongside this, X-ray µCT techniques were developed in order to detect biofilms inside CVCs. Various contrast agent stains were evaluated using energy-dispersive X-ray spectroscopy (EDS) to further optimize these methods. Catheter material and biofilm were segmented using a semi-automated matlab script and quantified using the Avizo Fire software package. X-ray µCT was capable of distinguishing between the degree of biofilm formation across different segments of a CVC flow model. EDS screening of single- and dual-compound contrast stains identified 10 nm gold and silver nitrate as the optimum contrast agent for X-ray µCT. This optimized method was then demonstrated to be capable of quantifying biofilms in an in vitro static biofilm formation model, with a strong correlation between biofilm detection via SEM and culture. X-ray µCT has good potential as a direct, non-invasive, non-destructive technology to image biofilms in CVCs, as well as other in vivo medical components in which biofilms accumulate in concealed areas.

Imaging approach to mechanistic study of nanoparticle interactions with the blood-brain barrier.

PubMed

Bramini, Mattia; Ye, Dong; Hallerbach, Anna; Nic Raghnaill, Michelle; Salvati, Anna; Aberg, Christoffer; Dawson, Kenneth A

2014-05-27

Understanding nanoparticle interactions with the central nervous system, in particular the blood-brain barrier, is key to advances in therapeutics, as well as assessing the safety of nanoparticles. Challenges in achieving insights have been significant, even for relatively simple models. Here we use a combination of live cell imaging and computational analysis to directly study nanoparticle translocation across a human in vitro blood-brain barrier model. This approach allows us to identify and avoid problems in more conventional inferential in vitro measurements by identifying the catalogue of events of barrier internalization and translocation as they occur. Potentially this approach opens up the window of applicability of in vitro models, thereby enabling in depth mechanistic studies in the future. Model nanoparticles are used to illustrate the method. For those, we find that translocation, though rare, appears to take place. On the other hand, barrier uptake is efficient, and since barrier export is small, there is significant accumulation within the barrier.

Risk-Based High-Throughput Chemical Screening and Prioritization using Exposure Models and in Vitro Bioactivity Assays.

PubMed

Shin, Hyeong-Moo; Ernstoff, Alexi; Arnot, Jon A; Wetmore, Barbara A; Csiszar, Susan A; Fantke, Peter; Zhang, Xianming; McKone, Thomas E; Jolliet, Olivier; Bennett, Deborah H

2015-06-02

We present a risk-based high-throughput screening (HTS) method to identify chemicals for potential health concerns or for which additional information is needed. The method is applied to 180 organic chemicals as a case study. We first obtain information on how the chemical is used and identify relevant use scenarios (e.g., dermal application, indoor emissions). For each chemical and use scenario, exposure models are then used to calculate a chemical intake fraction, or a product intake fraction, accounting for chemical properties and the exposed population. We then combine these intake fractions with use scenario-specific estimates of chemical quantity to calculate daily intake rates (iR; mg/kg/day). These intake rates are compared to oral equivalent doses (OED; mg/kg/day), calculated from a suite of ToxCast in vitro bioactivity assays using in vitro-to-in vivo extrapolation and reverse dosimetry. Bioactivity quotients (BQs) are calculated as iR/OED to obtain estimates of potential impact associated with each relevant use scenario. Of the 180 chemicals considered, 38 had maximum iRs exceeding minimum OEDs (i.e., BQs > 1). For most of these compounds, exposures are associated with direct intake, food/oral contact, or dermal exposure. The method provides high-throughput estimates of exposure and important input for decision makers to identify chemicals of concern for further evaluation with additional information or more refined models.

In vitro dissolution method fitted to in vivo absorption profile of rivaroxaban immediate-release tablets applying in silico data.

PubMed

Wingert, Nathalie R; Dos Santos, Natália O; Campanharo, Sarah C; Simon, Elisa S; Volpato, Nadia M; Steppe, Martin

2018-05-01

This study aimed to develop and validate an in vitro dissolution method based on in silico-in vivo data to determine whether an in vitro-in vivo relationship could be established for rivaroxaban in immediate-release tablets. Oral drugs with high permeability but poorly soluble in aqueous media, such as the anticoagulant rivaroxaban, have a major potential to reach a high level of in vitro-in vivo relationship. Currently, there is no study on scientific literature approaching the development of RIV dissolution profile based on its in vivo performance. Drug plasma concentration values were modeled using computer simulation with adjustment of pharmacokinetic properties. Those values were converted into drug fractions absorbed by the Wagner-Nelson deconvolution approach. Gradual and continuous dissolution of RIV tablets was obtained with a 30 rpm basket on 50 mM sodium acetate +0.2% SDS, pH 6.5 medium. Dissolution was conducted for up to 180 min. The fraction absorbed was plotted against the drug fraction dissolved, and a linear point-to-point regression (R 2  = 0.9961) obtained. The in vitro dissolution method designed promoted a more convenient dissolution profile of RIV tablets, whereas it suggests a better relationship with in vivo performance.

In Vitro Drug Interaction Modeling of Combinations of Azoles with Terbinafine against Clinical Scedosporium prolificans Isolates

PubMed Central

Meletiadis, Joseph; Mouton, Johan W.; Meis, Jacques F. G. M.; Verweij, Paul E.

2003-01-01

The in vitro interaction between terbinafine and the azoles voriconazole, miconazole, and itraconazole against five clinical Scedosporium prolificans isolates after 48 and 72 h of incubation was tested by a microdilution checkerboard (eight-by-twelve) technique. The antifungal effects of the drugs alone and in combination on the fungal biomass as well as on the metabolic activity of fungi were measured using a spectrophotometric method and two colorimetric methods, based on the lowest drug concentrations showed 75 and 50% growth inhibition (MIC-1 and MIC-2, respectively). The nature and the intensity of the interactions were assessed using a nonparametric approach (fractional inhibitory concentration [FIC] index model) and a fully parametric response surface approach (Greco model) of the Loewe additivity (LA) no-interaction theory as well as a nonparametric (Prichard model) and a semiparametric response surface approaches of the Bliss independence (BI) no-interaction theory. Statistically significant synergy was found between each of the three azoles and terbinafine in all cases, although with different intensities. A 27- to 64-fold and 16- to 90-fold reduction of the geometric mean of the azole and terbinafine MICs, respectively, was observed when they were combined, resulting in FIC indices of <1 to 0.02. Using the MIC-1 higher levels of synergy were obtained, , which were more consistent between the two incubation periods than using the MIC-2. The strongest synergy among the azoles was found with miconazole using the BI-based models and with voriconazole using the LA-based models. The synergistic effects both on fungal growth and metabolic activity were more potent after 72 h of incubation. Fully parametric approaches in combination with the modified colorimetric method might prove useful for testing the in vitro interaction of antifungal drugs against filamentous fungi. PMID:12499177

Preventing hypothermia: comparison of current devices used by the US Army in an in vitro warmed fluid model.

PubMed

Allen, Paul B; Salyer, Steven W; Dubick, Michael A; Holcomb, John B; Blackbourne, Lorne H

2010-07-01

The purpose of this study was to develop an in vitro torso model constructed with fluid bags and to determine whether this model could be used to differentiate between the heat prevention performance of devices with active chemical or radiant forced-air heating systems compared with passive heat loss prevention devices. We tested three active (Hypothermia Prevention Management Kit [HPMK], Ready-Heat, and Bair Hugger) and five passive (wool, space blankets, Blizzard blankets, human remains pouch, and Hot Pocket) hypothermia prevention products. Active warming devices included products with chemically or electrically heated systems. Both groups were tested on a fluid model warmed to 37 degrees C versus a control with no warming device. Core temperatures were recorded every 5 minutes for 120 minutes in total. Products that prevent heat loss with an actively heated element performed better than most passive prevention methods. The original HPMK achieved and maintained significantly higher temperatures than all other methods and the controls at 120 minutes (p < 0.05). None of the devices with an actively heated element achieved the sustained 44 degrees C that could damage human tissue if left in place for 6 hours. The best passive methods of heat loss prevention were the Hot Pocket and Blizzard blanket, which performed the same as two of the three active heating methods tested at 120 minutes. Our in vitro fluid bag "torso" model seemed sensitive to detect heat loss in the evaluation of several active or passive warming devices. All active and most passive devices were better than wool blankets. Under conditions near room temperature, passive warming methods (Blizzard blanket or the Hot Pocket) were as effective as active warming devices other than the original HPMK. Further studies are necessary to determine how these data can translate to field conditions in preventing heat loss in combat casualties.

Dissolution and dissolution/permeation experiments for predicting systemic exposure following oral administration of the BCS class II drug clarithromycin.

PubMed

Kristin, Forner; René, Holm; Boontida, Morakul; Buraphacheep, Junyaprasert Varaporn; Maximilian, Ackermann; Johanna, Mazur; Peter, Langguth

2017-04-01

In order to save time and resources in early drug development, in vitro methods that correctly predict the formulation effect on oral drug absorption are necessary. The aim of this study was to 1) evaluate various BCS class II drug formulations with in vitro methods and in vivo in order to 2) determine which in vitro method best correlates with the in vivo results. Clarithromycin served as model compound in formulations with different particle sizes and content of excipients. The performed in vitro experiments were dissolution and dissolution/permeation experiments across two types of membrane, Caco-2 cells and excised rat intestinal sheets. The in vivo study was performed in rats. The oral absorption was enhanced by downsizing drug particles and by increasing the excipient concentration. This correlated strongly with the flux across Caco-2 cells but not with the other in vitro experiments. The insufficient correlation with the dissolution experiments can be partly explained by excipient caused problems during the filtration step. The very poor correlation of the in vivo data with the flux across excised rat intestinal sheets might be due to an artificially enlarged mucus layer ex vivo. In conclusion, downsizing BCS class II drug particles and the addition of surfactants enhanced the in vivo absorption, which was best depicted by dissolution/permeation experiments across Caco-2 cells. This setup is proposed as best model to predict the in vivo formulation effect. Also, this is the first study to evaluate the impact of the nature of the permeation membrane in dissolution/permeation experiments. Copyright © 2017 Elsevier B.V. All rights reserved.

Ex vivo rabbit and human corneas as models for bacterial and fungal keratitis.

PubMed

Pinnock, Abigail; Shivshetty, Nagaveni; Roy, Sanhita; Rimmer, Stephen; Douglas, Ian; MacNeil, Sheila; Garg, Prashant

2017-02-01

In the study of microbial keratitis, in vivo animal models often require a large number of animals, and in vitro monolayer cell culture does not maintain the three-dimensional structure of the tissues or cell-to-cell communication of in vivo models. Here, we propose reproducible ex vivo models of single- and dual-infection keratitis as an alternative to in vivo and in vitro models. Excised rabbit and human corneoscleral rims maintained in organ culture were infected using 10 8 cells of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans or Fusarium solani. The infection was introduced by wounding with a scalpel and exposing corneas to the microbial suspension or by intrastromal injection. Post-inoculation, corneas were maintained for 24 and 48 h at 37 °C. After incubation, corneas were either homogenised to determine colony-forming units (CFU)/cornea or processed for histological examination using routine staining methods. Single- and mixed-species infections were compared. We observed a significant increase in CFU after 48 h compared to 24 h with S. aureus and P. aeruginosa. However, no such increase was observed in corneas infected with C. albicans or F. solani. The injection method yielded an approximately two- to 100-fold increase (p < 0.05) in the majority of organisms from infected corneas. Histology of the scalpel-wounded and injection models indicated extensive infiltration of P. aeruginosa throughout the entire cornea, with less infiltration observed for S. aureus, C. albicans and F. solani. The models also supported dual infections. Both scalpel wounding and injection methods are suitable for inducing infection of ex vivo rabbit and human cornea models. These simple and reproducible models will be useful as an alternative to in vitro and in vivo models for investigating the detection and treatment of microbial keratitis, particularly when this might be due to two infective organisms.

Effect of Nanoparticles on Complement System in Cell Culture Model

DTIC Science & Technology

2006-09-15

case complement activation considerably differs between nanoparticles , being the highest in case of fullerene, ferric oxide and aluminium oxide ... oxide (CdO; 1 µm), manganese oxide (MnO2; 1-2 µm), and tungsten (W; 27 µm) were assessed. Additionally the effects of nanoparticles coated with...using in vitro system. Obtained results indicate that: 1. Nanoparticles toxicity in vitro can’t be measured using methods which were designed

Comparison of two different in vivo models and an in vitro model for caloric determination of four novel fiber ingredients.

PubMed

Cervantes-Pahm, Sarah; Knapp, Brenda K; Kim, Beob G; Liu, Yanhong; Parsons, Carl M; Fahey, George C; Stein, Hans H

2013-12-18

The objective of this study was to compare two in vivo methods using pigs and roosters and an in vitro method for determining the caloric value of four fiber sources [i.e., two resistant starches (RS 60 and RS 75), soluble corn fiber (SCF 70), and pullulan]. Metabolizable energy (ME) in pigs and true metabolizable energy (TMEn) in roosters were determined by using 72 barrows and 24 roosters, respectively. A two-step in vitro procedure was used to quantify monosaccharides released. Results of the two in vivo experiments corresponded well with RS 75 having the least caloric value (7.55 MJ/kg in pigs; 6.19 MJ/kg in roosters) and pullulan having the greatest caloric value (12.21 MJ/kg in pigs; 13.94 MJ/kg in roosters). The caloric values for all the fiber ingredients were less (P < 0.05) than in MD both in pigs and in roosters. Despite some limitations, results of the in vitro procedure corresponded well with the in vivo experiments where the concentration of glucose hydrolyzed from RS 60, RS 75, and SCF 70, but not pullulan, was less (P < 0.05) than the concentration of glucose hydrolyzed from MD. However, the greatest accuracy was obtained in the in vivo experiments.

Cell cultures in drug discovery and development: The need of reliable in vitro-in vivo extrapolation for pharmacodynamics and pharmacokinetics assessment.

PubMed

Jaroch, Karol; Jaroch, Alina; Bojko, Barbara

2018-01-05

For ethical and cost-related reasons, use of animals for the assessment of mode of action, metabolism and/or toxicity of new drug candidates has been increasingly scrutinized in research and industrial applications. Implementation of the 3 "Rs" 1 ; rule (Reduction, Replacement, Refinement) through development of in silico or in vitro assays has become an essential element of risk assessment. Physiologically based pharmacokinetic (PBPK 2 ) modeling is the most potent in silico tool used for extrapolation of pharmacokinetic parameters to animal or human models from results obtained in vitro. Although, many types of in vitro assays are conducted during drug development, use of cell cultures is the most reliable one. Two-dimensional (2D) cell cultures have been a part of drug development for many years. Nowadays, their role is decreasing in favor of three-dimensional (3D) cell cultures and co-cultures. 3D cultures exhibit protein expression patterns and intercellular junctions that are closer to in vivo states in comparison to classical monolayer cultures. Co-cultures allow for examinations of the mutual influence of different cell lines. However, the complexity and high costs of co-cultures and 3D equipment exclude such methods from high-throughput screening (HTS). 3 In vitro absorption, distribution, metabolism, and excretion assessment, as well as drug-drug interaction (DDI), are usually performed with the use of various cell culture based assays. Progress in in silico and in vitro methods can lead to better in vitro-in vivo extrapolation (IVIVE 4 ) outcomes and have a potential to contribute towards a significant reduction in the number of laboratory animals needed for drug research. As such, concentrated efforts need to be spent towards the development of an HTS in vitro platform with satisfactory IVIVE features. Copyright © 2017 Elsevier B.V. All rights reserved.

In vitro generation of three-dimensional substrate-adherent embryonic stem cell-derived neural aggregates for application in animal models of neurological disorders.

PubMed

Hargus, Gunnar; Cui, Yi-Fang; Dihné, Marcel; Bernreuther, Christian; Schachner, Melitta

2012-05-01

In vitro-differentiated embryonic stem (ES) cells comprise a useful source for cell replacement therapy, but the efficiency and safety of a translational approach are highly dependent on optimized protocols for directed differentiation of ES cells into the desired cell types in vitro. Furthermore, the transplantation of three-dimensional ES cell-derived structures instead of a single-cell suspension may improve graft survival and function by providing a beneficial microenvironment for implanted cells. To this end, we have developed a new method to efficiently differentiate mouse ES cells into neural aggregates that consist predominantly (>90%) of postmitotic neurons, neural progenitor cells, and radial glia-like cells. When transplanted into the excitotoxically lesioned striatum of adult mice, these substrate-adherent embryonic stem cell-derived neural aggregates (SENAs) showed significant advantages over transplanted single-cell suspensions of ES cell-derived neural cells, including improved survival of GABAergic neurons, increased cell migration, and significantly decreased risk of teratoma formation. Furthermore, SENAs mediated functional improvement after transplantation into animal models of Parkinson's disease and spinal cord injury. This unit describes in detail how SENAs are efficiently derived from mouse ES cells in vitro and how SENAs are isolated for transplantation. Furthermore, methods are presented for successful implantation of SENAs into animal models of Huntington's disease, Parkinson's disease, and spinal cord injury to study the effects of stem cell-derived neural aggregates in a disease context in vivo.

Effects of varying dietary ratios of corn silage to alfalfa silage on digestion of neutral detergent fiber in lactating dairy cows.

PubMed

Lopes, F; Cook, D E; Combs, D K

2015-09-01

An in vivo study was performed to test an in vitro procedure and model that predicts total-tract neutral detergent fiber (NDF) digestibility for lactating dairy cattle. Corn silage (CS) and alfalfa silage (AS) were used as forages for this study. These forages had similar NDF composition, but fiber in the CS contained less indigestible NDF compared with AS (35.5 and 47.8% of indigestible NDF, respectively). The in vitro method estimated rate of digestion of alfalfa potentially digestible NDF to be approximately 2 times faster than CS fiber (6.11 and 3.21%/h, respectively). Four diets were formulated containing different proportions of CS to AS: 100CS:0AS, 67CS:33AS, 33CS:67AS, and 0CS:100AS, as percentage of diet DM basis. The objective was to construct diets that contained approximately similar levels of NDF but with different pool sizes and rates of digestion of potentially digestible NDF. Diets were fed to 8 ruminally cannulated, multiparous, lactating dairy cows in a replicated 4×4 Latin square with 21-d periods. Total-tract fiber digestibility and fiber digestion kinetic parameters observed in vivo were compared with the values predicted by the in vitro assay and model. Total-tract NDF digestibility coefficients were similar (41.8 and 40.6% of total NDF) for the in vitro and in vivo methods, respectively. As the proportion of dietary alfalfa increased, the digestibility of NDF increased. The rate of digestion of potentially digestible NDF predicted from the in vitro assay was also similar to what was observed in vivo. Results suggest that the in vitro total-tract NDF digestibility model could be used to predict rate of fiber digestion and NDF digestibility for lactating dairy cattle. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Mathematical Modeling and Optimizing of in Vitro Hormonal Combination for G × N15 Vegetative Rootstock Proliferation Using Artificial Neural Network-Genetic Algorithm (ANN-GA)

PubMed Central

Arab, Mohammad M.; Yadollahi, Abbas; Ahmadi, Hamed; Eftekhari, Maliheh; Maleki, Masoud

2017-01-01

The efficiency of a hybrid systems method which combined artificial neural networks (ANNs) as a modeling tool and genetic algorithms (GAs) as an optimizing method for input variables used in ANN modeling was assessed. Hence, as a new technique, it was applied for the prediction and optimization of the plant hormones concentrations and combinations for in vitro proliferation of Garnem (G × N15) rootstock as a case study. Optimizing hormones combination was surveyed by modeling the effects of various concentrations of cytokinin–auxin, i.e., BAP, KIN, TDZ, IBA, and NAA combinations (inputs) on four growth parameters (outputs), i.e., micro-shoots number per explant, length of micro-shoots, developed callus weight (CW) and the quality index (QI) of plantlets. Calculation of statistical values such as R2 (coefficient of determination) related to the accuracy of ANN-GA models showed a considerably higher prediction accuracy for ANN models, i.e., micro-shoots number: R2 = 0.81, length of micro-shoots: R2 = 0.87, CW: R2 = 0.88, QI: R2 = 0.87. According to the results, among the input variables, BAP (19.3), KIN (9.64), and IBA (2.63) showed the highest values of variable sensitivity ratio for proliferation rate. The GA showed that media containing 1.02 mg/l BAP in combination with 0.098 mg/l IBA could lead to the optimal proliferation rate (10.53) for G × N15 rootstock. Another objective of the present study was to compare the performance of predicted and optimized cytokinin–auxin combination with the best optimized obtained concentrations of our other experiments. Considering three growth parameters (length of micro-shoots, micro-shoots number, and proliferation rate), the last treatment was found to be superior to the rest of treatments for G × N15 rootstock in vitro multiplication. Very little difference between the ANN predicted and experimental data confirmed high capability of ANN-GA method in predicting new optimized protocols for plant in vitro propagation. PMID:29163583

The effectiveness of adhesives on the retention of mandibular free end saddle partial dentures: An in vitro study.

PubMed

Quiney, Daniel; Nishio Ayre, Wayne; Milward, Paul

2017-07-01

Existing in vitro methods for testing denture adhesives do not fully replicate the complex oral geometries and environment; and in vivo methods are qualitative, prone to bias and not easily reproducible. The purpose of this study was to develop a novel, quantitative and more accurate model to test the effect of adhesives on the retentive force of mandibular free end saddle partial dentures. An in vitro model was developed based on an anatomically accurate cast of a clinical case. Experimentally, the amount of adhesive was varied (0.2g-1g) and the tensile force required for displacement was measured. Different commercially available adhesives were then tested at the optimum volume using the in vitro model. A 3D finite element model of the denture was used to assess how the forces to induce denture displacement varied according to the position of the force along the saddle length. The mass of adhesive was found to significantly alter retention forces, with 0.4-0.7g being the optimum range for this particular scenario. Use of adhesives significantly improved mandibular free end saddle partial denture retention with the worst performing adhesive increasing retention nine-fold whilst the best performing adhesive increased retention twenty three-fold. The finite element model revealed that 77% more force was required to displace the denture by positioning forces towards the mesial end of the saddle compared to the distal end. An in vitro denture adhesive model was developed, which demonstrated that mass of adhesive plays a significant role in enhancing denture retention and supported the design principle of placing as few teeth as clinically necessary on the distal end of the free end saddles. Limiting the position of teeth on free end saddles to the mesial and mid portion of the saddle will reduce displacements caused by mastication. The movement of mandibular free end saddle partial dentures can be restricted with the use of denture adhesives. Altering the mass of adhesive used can further improve the retention of mandibular free end saddle partial dentures for patients. Copyright © 2017 Elsevier Ltd. All rights reserved.

Probing amyloid protein aggregation with optical superresolution methods: from the test tube to models of disease

PubMed Central

Kaminski, Clemens F.; Kaminski Schierle, Gabriele S.

2016-01-01

Abstract. The misfolding and self-assembly of intrinsically disordered proteins into insoluble amyloid structures are central to many neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Optical imaging of this self-assembly process in vitro and in cells is revolutionizing our understanding of the molecular mechanisms behind these devastating conditions. In contrast to conventional biophysical methods, optical imaging and, in particular, optical superresolution imaging, permits the dynamic investigation of the molecular self-assembly process in vitro and in cells, at molecular-level resolution. In this article, current state-of-the-art imaging methods are reviewed and discussed in the context of research into neurodegeneration. PMID:27413767

The Development and Validation of an In Vitro Airway Model to Assess Realistic Airway Deposition and Drug Permeation Behavior of Orally Inhaled Products Across Synthetic Membranes.

PubMed

Huynh, Bao K; Traini, Daniela; Farkas, Dale R; Longest, P Worth; Hindle, Michael; Young, Paul M

2018-04-01

Current in vitro approaches to assess lung deposition, dissolution, and cellular transport behavior of orally inhaled products (OIPs) have relied on compendial impactors to collect drug particles that are likely to deposit in the airway; however, the main drawback with this approach is that these impactors do not reflect the airway and may not necessarily represent drug deposition behavior in vivo. The aim of this article is to describe the development and method validation of a novel hybrid in vitro approach to assess drug deposition and permeation behavior in a more representative airway model. The medium-sized Virginia Commonwealth University (VCU) mouth-throat (MT) and tracheal-bronchial (TB) realistic upper airway models were used in this study as representative models of the upper airway. The TB model was modified to accommodate two Snapwell ® inserts above the first TB airway bifurcation region to collect deposited nebulized ciprofloxacin-hydrochloride (CIP-HCL) droplets as a model drug aerosol system. Permeation characteristics of deposited nebulized CIP-HCL droplets were assessed across different synthetic membranes using the Snapwell test system. The Snapwell test system demonstrated reproducible and discriminatory drug permeation profiles for already dissolved and nebulized CIP-HCL droplets through a range of synthetic permeable membranes under different test conditions. The rate and extent of drug permeation depended on the permeable membrane material used, presence of a stirrer in the receptor compartment, and, most importantly, the drug collection method. This novel hybrid in vitro approach, which incorporates a modified version of a realistic upper airway model, coupled with the Snapwell test system holds great potential to evaluate postairway deposition characteristics, such as drug permeation and particle dissolution behavior of OIPs. Future studies will expand this approach using a cell culture-based setup instead of synthetic membranes, within a humidified chamber, to assess airway epithelia transport behavior in a more representative manner.

Insights into an Optimization of Plasmodium vivax Sal-1 In Vitro Culture: The Aotus Primate Model.

PubMed

Shaw-Saliba, Kathryn; Thomson-Luque, Richard; Obaldía, Nicanor; Nuñez, Marlon; Dutary, Sahir; Lim, Caeul; Barnes, Samantha; Kocken, Clemens H M; Duraisingh, Manoj T; Adams, John H; Pasini, Erica M

2016-07-01

Malaria is one of the most significant tropical diseases, and of the Plasmodium species that cause human malaria, P. vivax is the most geographically widespread. However, P. vivax remains a relatively neglected human parasite since research is typically limited to laboratories with direct access to parasite isolates from endemic field settings or from non-human primate models. This restricted research capacity is in large part due to the lack of a continuous P. vivax in vitro culture system, which has hampered the ability for experimental research needed to gain biological knowledge and develop new therapies. Consequently, efforts to establish a long-term P. vivax culture system are confounded by our poor knowledge of the preferred host cell and essential nutrients needed for in vitro propagation. Reliance on very heterogeneous P. vivax field isolates makes it difficult to benchmark parasite characteristics and further complicates development of a robust and reliable culture method. In an effort to eliminate parasite variability as a complication, we used a well-defined Aotus-adapted P. vivax Sal-1 strain to empirically evaluate different short-term in vitro culture conditions and compare them with previous reported attempts at P. vivax in vitro culture Most importantly, we suggest that reticulocyte enrichment methods affect invasion efficiency and we identify stabilized forms of nutrients that appear beneficial for parasite growth, indicating that P. vivax may be extremely sensitive to waste products. Leuko-depletion methods did not significantly affect parasite development. Formatting changes such as shaking and static cultures did not seem to have a major impact while; in contrast, the starting haematocrit affected both parasite invasion and growth. These results support the continued use of Aotus-adapted Sal-1 for development of P. vivax laboratory methods; however, further experiments are needed to optimize culture conditions to support long-term parasite development.

Insights into an Optimization of Plasmodium vivax Sal-1 In Vitro Culture: The Aotus Primate Model

PubMed Central

Obaldía, Nicanor; Nuñez, Marlon; Dutary, Sahir; Lim, Caeul; Barnes, Samantha; Kocken, Clemens H. M.; Duraisingh, Manoj T.; Adams, John H.; Pasini, Erica M.

2016-01-01

Malaria is one of the most significant tropical diseases, and of the Plasmodium species that cause human malaria, P. vivax is the most geographically widespread. However, P. vivax remains a relatively neglected human parasite since research is typically limited to laboratories with direct access to parasite isolates from endemic field settings or from non-human primate models. This restricted research capacity is in large part due to the lack of a continuous P. vivax in vitro culture system, which has hampered the ability for experimental research needed to gain biological knowledge and develop new therapies. Consequently, efforts to establish a long-term P. vivax culture system are confounded by our poor knowledge of the preferred host cell and essential nutrients needed for in vitro propagation. Reliance on very heterogeneous P. vivax field isolates makes it difficult to benchmark parasite characteristics and further complicates development of a robust and reliable culture method. In an effort to eliminate parasite variability as a complication, we used a well-defined Aotus-adapted P. vivax Sal-1 strain to empirically evaluate different short-term in vitro culture conditions and compare them with previous reported attempts at P. vivax in vitro culture Most importantly, we suggest that reticulocyte enrichment methods affect invasion efficiency and we identify stabilized forms of nutrients that appear beneficial for parasite growth, indicating that P. vivax may be extremely sensitive to waste products. Leuko-depletion methods did not significantly affect parasite development. Formatting changes such as shaking and static cultures did not seem to have a major impact while; in contrast, the starting haematocrit affected both parasite invasion and growth. These results support the continued use of Aotus-adapted Sal-1 for development of P. vivax laboratory methods; however, further experiments are needed to optimize culture conditions to support long-term parasite development. PMID:27463518

Comparison of in vitro and in situ methods in evaluation of forage digestibility in ruminants.

PubMed

Krizsan, S J; Nyholm, L; Nousiainen, J; Südekum, K-H; Huhtanen, P

2012-09-01

The objective of this study was to compare the application of different in vitro and in situ methods in empirical and mechanistic predictions of in vivo OM digestibility (OMD) and their associations to near-infrared reflectance spectroscopy spectra for a variety of forages. Apparent in vivo OMD of silages made from alfalfa (n = 2), corn (n = 9), corn stover (n = 2), grass (n = 11), whole crops of wheat and barley (n = 8) and red clover (n = 7), and fresh alfalfa (n = 1), grass hays (n = 5), and wheat straws (n = 5) had previously been determined in sheep. Concentrations of indigestible NDF (iNDF) in all forage samples were determined by a 288-h ruminal in situ incubation. Gas production of isolated forage NDF was measured by in vitro incubations for 72 h. In vitro pepsin-cellulase OM solubility (OMS) of the forages was determined by a 2-step gravimetric digestion method. Samples were also subjected to a 2-step determination of in vitro OMD based on buffered rumen fluid and pepsin. Further, rumen fluid digestible OM was determined from a single 96-h incubation at 38°C. Digestibility of OM from the in situ and the in vitro incubations was calculated according to published empirical equations, which were either forage specific or general (1 equation for all forages) within method. Indigestible NDF was also used in a mechanistic model to predict OMD. Predictions of OMD were evaluated by residual analysis using the GLM procedure in SAS. In vitro OMS in a general prediction equation of OMD did not display a significant forage-type effect on the residuals (observed - predicted OMD; P = 0.10). Predictions of OMD within forage types were consistent between iNDF and the 2-step in vitro method based on rumen fluid. Root mean square error of OMD was least (0.032) when the prediction was based on a general forage equation of OMS. However, regenerating a simple regression for iNDF by omitting alfalfa and wheat straw reduced the root mean square error of OMD to 0.025. Indigestible NDF in a general forage equation predicted OMD without any bias (P ≥ 0.16), and root mean square error of prediction was smallest among all methods when alfalfa and wheat straw samples were excluded. Our study suggests that compared with the in vitro laboratory methods, iNDF used in forage-specific equations will improve overall predictions of forage in vivo OMD. The in vitro and in situ methods performed equally well in calibrations of iNDF or OMD by near-infrared reflectance spectroscopy.

Measured and Modeled Toxicokinetics in Cultured Fish Cells and Application to In Vitro - In Vivo Toxicity Extrapolation

PubMed Central

Stadnicka-Michalak, Julita; Tanneberger, Katrin; Schirmer, Kristin; Ashauer, Roman

2014-01-01

Effect concentrations in the toxicity assessment of chemicals with fish and fish cells are generally based on external exposure concentrations. External concentrations as dose metrics, may, however, hamper interpretation and extrapolation of toxicological effects because it is the internal concentration that gives rise to the biological effective dose. Thus, we need to understand the relationship between the external and internal concentrations of chemicals. The objectives of this study were to: (i) elucidate the time-course of the concentration of chemicals with a wide range of physicochemical properties in the compartments of an in vitro test system, (ii) derive a predictive model for toxicokinetics in the in vitro test system, (iii) test the hypothesis that internal effect concentrations in fish (in vivo) and fish cell lines (in vitro) correlate, and (iv) develop a quantitative in vitro to in vivo toxicity extrapolation method for fish acute toxicity. To achieve these goals, time-dependent amounts of organic chemicals were measured in medium, cells (RTgill-W1) and the plastic of exposure wells. Then, the relation between uptake, elimination rate constants, and log KOW was investigated for cells in order to develop a toxicokinetic model. This model was used to predict internal effect concentrations in cells, which were compared with internal effect concentrations in fish gills predicted by a Physiologically Based Toxicokinetic model. Our model could predict concentrations of non-volatile organic chemicals with log KOW between 0.5 and 7 in cells. The correlation of the log ratio of internal effect concentrations in fish gills and the fish gill cell line with the log KOW was significant (r>0.85, p = 0.0008, F-test). This ratio can be predicted from the log KOW of the chemical (77% of variance explained), comprising a promising model to predict lethal effects on fish based on in vitro data. PMID:24647349

Saccharomyces cerevisiae show low levels of traversal across human endothelial barrier in vitro.

PubMed

Pérez-Torrado, Roberto; Querol, Amparo

2017-01-01

Background :   Saccharomyces cerevisiae is generally considered safe, and is involved in the production of many types of foods and dietary supplements. However, some isolates, which are genetically related to strains used in brewing and baking, have shown virulent traits, being able to produce infections in humans, mainly in immunodeficient patients. This can lead to systemic infections in humans. Methods : In this work, we studied S. cerevisiae isolates in an in vitro human endothelial barrier model, comparing their behaviour with that of several strains of the related pathogens Candida glabrata and Candida albicans . Results : The results showed that this food related yeast is able to cross the endothelial barrier in vitro . However, in contrast to C. glabrata and C. albicans , S. cerevisiae showed very low levels of traversal. Conclusions : We conclude that using an in vitro human endothelial barrier model with S. cerevisiae can be useful to evaluate the safety of S. cerevisiae strains isolated from foods.

[Preparation and in vitro release characteristics of vincristine sulphate loaded poly (butylcyanoacrylate) nanoparticles].

PubMed

Tan, Rong; Liu, Ying; Feng, Nianping; Zhao, Jihui

2011-06-01

To prepare vincristine sulphate loaded poly (butylcyanoacrylate) nanoparticles (VCR-PBCA-NPs) and to investigate the in vitro release charactersitics. VCR-PBCA-NPs were prepared by emulsion polymerization method, and characterized for morphology, particle size, drug encapsulation efficiency and loading efficiency. The formulation was optimized using central composite design and response surface methodology. In vitro release study of VCR-PBCA-NPs was performed by dialysis technique. Model fitting was used to determine the kinetics and to discuss the mechanism. The nanoparticles were spherical and uniform with a mean diameter of (98.9 +/- 3.05) nm. The drug encapsulation efficiency and loading efficiency were (55.23 +/- 0.96)% and (7.87 +/- 0.11)%, respectively. In vitro release results showed that 63.66% of VCR was released from VCR-PBCA-NPs in 4 h, and the Weibull model fitted VCR release pattern best. The VCR-PBCA-NPs prepared in this study showed sustained release compared with VCR solution.

Solid lipid nanoparticles as carrier for sunscreens: in vitro release and in vivo skin penetration.

PubMed

Wissing, S A; Müller, R H

2002-06-17

The aim of this study was the comparison of two different formulations (solid lipid nanoparticles (SLN) and conventional o/w emulsion) as carrier systems for the molecular sunscreen oxybenzone. The influence of the carrier on the rate of release was studied in vitro with a membrane-free model. The release rate could be decreased by up to 50% with the SLN formulation. Further in vitro measurements with static Franz diffusion cells were performed. In vivo, penetration of oxybenzone into stratum corneum on the forearm was investigated by the tape stripping method. It was shown that the rate of release is strongly dependent upon the formulation and could be decreased by 30-60% in SLN formulations. In all test models, oxybenzone was released and penetrated into human skin more quickly and to a greater extent from the emulsions. The rate of release also depends upon the total concentration of oxybenzone in the formulation. In vitro-in vivo correlations could be made qualitatively.

A comparison of quantitative methods for clinical imaging with hyperpolarized (13)C-pyruvate.

PubMed

Daniels, Charlie J; McLean, Mary A; Schulte, Rolf F; Robb, Fraser J; Gill, Andrew B; McGlashan, Nicholas; Graves, Martin J; Schwaiger, Markus; Lomas, David J; Brindle, Kevin M; Gallagher, Ferdia A

2016-04-01

Dissolution dynamic nuclear polarization (DNP) enables the metabolism of hyperpolarized (13)C-labelled molecules, such as the conversion of [1-(13)C]pyruvate to [1-(13)C]lactate, to be dynamically and non-invasively imaged in tissue. Imaging of this exchange reaction in animal models has been shown to detect early treatment response and correlate with tumour grade. The first human DNP study has recently been completed, and, for widespread clinical translation, simple and reliable methods are necessary to accurately probe the reaction in patients. However, there is currently no consensus on the most appropriate method to quantify this exchange reaction. In this study, an in vitro system was used to compare several kinetic models, as well as simple model-free methods. Experiments were performed using a clinical hyperpolarizer, a human 3 T MR system, and spectroscopic imaging sequences. The quantitative methods were compared in vivo by using subcutaneous breast tumours in rats to examine the effect of pyruvate inflow. The two-way kinetic model was the most accurate method for characterizing the exchange reaction in vitro, and the incorporation of a Heaviside step inflow profile was best able to describe the in vivo data. The lactate time-to-peak and the lactate-to-pyruvate area under the curve ratio were simple model-free approaches that accurately represented the full reaction, with the time-to-peak method performing indistinguishably from the best kinetic model. Finally, extracting data from a single pixel was a robust and reliable surrogate of the whole region of interest. This work has identified appropriate quantitative methods for future work in the analysis of human hyperpolarized (13)C data. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.

Improved Predictions of Drug-Drug Interactions Mediated by Time-Dependent Inhibition of CYP3A.

PubMed

Yadav, Jaydeep; Korzekwa, Ken; Nagar, Swati

2018-05-07

Time-dependent inactivation (TDI) of cytochrome P450s (CYPs) is a leading cause of clinical drug-drug interactions (DDIs). Current methods tend to overpredict DDIs. In this study, a numerical approach was used to model complex CYP3A TDI in human-liver microsomes. The inhibitors evaluated included troleandomycin (TAO), erythromycin (ERY), verapamil (VER), and diltiazem (DTZ) along with the primary metabolites N-demethyl erythromycin (NDE), norverapamil (NV), and N-desmethyl diltiazem (NDD). The complexities incorporated into the models included multiple-binding kinetics, quasi-irreversible inactivation, sequential metabolism, inhibitor depletion, and membrane partitioning. The resulting inactivation parameters were incorporated into static in vitro-in vivo correlation (IVIVC) models to predict clinical DDIs. For 77 clinically observed DDIs, with a hepatic-CYP3A-synthesis-rate constant of 0.000 146 min -1 , the average fold difference between the observed and predicted DDIs was 3.17 for the standard replot method and 1.45 for the numerical method. Similar results were obtained using a synthesis-rate constant of 0.000 32 min -1 . These results suggest that numerical methods can successfully model complex in vitro TDI kinetics and that the resulting DDI predictions are more accurate than those obtained with the standard replot approach.

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier.

PubMed

Watson, P Marc D; Paterson, Judy C; Thom, George; Ginman, Ulrika; Lundquist, Stefan; Webster, Carl I

2013-06-18

Modelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies. Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting. Using a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS endothelial cell types can be obtained, and these can be passaged onto large numbers of cell culture inserts for in vitro permeability studies. The passaged brain and spinal cord endothelial cells are pure and express endothelial markers, tight junction proteins and intracellular transport machinery. Further, both models exhibit tight, functional barrier characteristics that are discriminating against large and small molecules in permeability assays and show functional expression of the pharmaceutically important P-gp efflux transporter. Our techniques allow the provision of high yields of robust sister cultures of endothelial cells that accurately model the blood-CNS barriers in vitro. These models are ideally suited for use in studying the biology of the blood-brain barrier and blood-spinal cord barrier in vitro and for pre-clinical drug discovery.

Mathematical modeling of HIV-like particle assembly in vitro.

PubMed

Liu, Yuewu; Zou, Xiufen

2017-06-01

In vitro, the recombinant HIV-1 Gag protein can generate spherical particles with a diameter of 25-30 nm in a fully defined system. It has approximately 80 building blocks, and its intermediates for assembly are abundant in geometry. Accordingly, there are a large number of nonlinear equations in the classical model. Therefore, it is difficult to compute values of geometry parameters for intermediates and make the mathematical analysis using the model. In this work, we develop a new model of HIV-like particle assembly in vitro by using six-fold symmetry of HIV-like particle assembly to decrease the number of geometry parameters. This method will greatly reduce computational costs and facilitate the application of the model. Then, we prove the existence and uniqueness of the positive equilibrium solution for this model with 79 nonlinear equations. Based on this model, we derive the interesting result that concentrations of all intermediates at equilibrium are independent of three important parameters, including two microscopic on-rate constants and the size of nucleating structure. Before equilibrium, these three parameters influence the concentration variation rates of all intermediates. We also analyze the relationship between the initial concentration of building blocks and concentrations of all intermediates. Furthermore, the bounds of concentrations of free building blocks and HIV-like particles are estimated. These results will be helpful to guide HIV-like particle assembly experiments and improve our understanding of the assembly dynamics of HIV-like particles in vitro. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation of anti-inflammatory potential of leaf extracts of Skimmia anquetilia

PubMed Central

Kumar, Vijender; Bhat, Zulfiqar Ali; Kumar, Dinesh; Khan, NA; Chashoo, IA

2012-01-01

Objective To evaluate anti-inflammatory potential of leaf extract of Skimmia anquetilia by in-vitro and in-vivo anti-inflammatory models. Methods Acute toxicity study was carried out to determine the toxicity level of different extract using acute toxic class method as described in Organization of Economic Co-operation and Development Guidelines No.423. Carrageenan (1% w/w) was administered and inflammation was induced in rat paw. The leaf extracts of Skimmia anquetilia were evaluated for anti-inflammatory activity by in-vitro human red blood cell (HRBC) membrane stabilization method and in-vivo carrangeenan-induced rat paw edema method. Results The in-vitro membrane stabilizing test showed petroleum ether (PE), chloroform (CE), ethyl acetate (EE), methanol (ME) and aqueous extracts (AE) showed 49.44%, 59.39%, 60.15%, 68.40% and 52.18 % protection, respectively as compared to control groups. The in-vivo results of CE, EE and ME showed 58.20%, 60.17% and 67.53% inhibition of inflammation after 6h administration of test drugs in albino rats. The potency of the leaf extracts of Skimmia anquetilia were compared with standard diclofenac (10 mg/kg) which showed 74.18% protection in in-vitro HRBC membrane stabilization test and 71.64% inhibition in in-vivo carrangeenan-induced rat paw edema model. The ME showed a dose dependent significant (P< 0.01) anti-inflammatory activity in human red blood cell membrane stabilization test and reduction of edema in carrageenan induced rat paw edema. Conclusions The present investigation has confirmed the anti-inflammatory activity of Skimmia anquetilia due to presence of bioactive phytoconstitutes for the first time and provide the pharmacological evidence in favor of traditional claim of Skimmia anquetilia as an anti- inflammatory agent. PMID:23569983

Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity.

PubMed

Clippinger, Amy J; Allen, David; Behrsing, Holger; BéruBé, Kelly A; Bolger, Michael B; Casey, Warren; DeLorme, Michael; Gaça, Marianna; Gehen, Sean C; Glover, Kyle; Hayden, Patrick; Hinderliter, Paul; Hotchkiss, Jon A; Iskandar, Anita; Keyser, Brian; Luettich, Karsta; Ma-Hock, Lan; Maione, Anna G; Makena, Patrudu; Melbourne, Jodie; Milchak, Lawrence; Ng, Sheung P; Paini, Alicia; Page, Kathryn; Patlewicz, Grace; Prieto, Pilar; Raabe, Hans; Reinke, Emily N; Roper, Clive; Rose, Jane; Sharma, Monita; Spoo, Wayne; Thorne, Peter S; Wilson, Daniel M; Jarabek, Annie M

2018-06-20

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substance's physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

A Novel Two-Step Hierarchical Quantitative Structure–Activity Relationship Modeling Work Flow for Predicting Acute Toxicity of Chemicals in Rodents

PubMed Central

Zhu, Hao; Ye, Lin; Richard, Ann; Golbraikh, Alexander; Wright, Fred A.; Rusyn, Ivan; Tropsha, Alexander

2009-01-01

Background Accurate prediction of in vivo toxicity from in vitro testing is a challenging problem. Large public–private consortia have been formed with the goal of improving chemical safety assessment by the means of high-throughput screening. Objective A wealth of available biological data requires new computational approaches to link chemical structure, in vitro data, and potential adverse health effects. Methods and results A database containing experimental cytotoxicity values for in vitro half-maximal inhibitory concentration (IC50) and in vivo rodent median lethal dose (LD50) for more than 300 chemicals was compiled by Zentralstelle zur Erfassung und Bewertung von Ersatz- und Ergaenzungsmethoden zum Tierversuch (ZEBET; National Center for Documentation and Evaluation of Alternative Methods to Animal Experiments). The application of conventional quantitative structure–activity relationship (QSAR) modeling approaches to predict mouse or rat acute LD50 values from chemical descriptors of ZEBET compounds yielded no statistically significant models. The analysis of these data showed no significant correlation between IC50 and LD50. However, a linear IC50 versus LD50 correlation could be established for a fraction of compounds. To capitalize on this observation, we developed a novel two-step modeling approach as follows. First, all chemicals are partitioned into two groups based on the relationship between IC50 and LD50 values: One group comprises compounds with linear IC50 versus LD50 relationships, and another group comprises the remaining compounds. Second, we built conventional binary classification QSAR models to predict the group affiliation based on chemical descriptors only. Third, we developed k-nearest neighbor continuous QSAR models for each subclass to predict LD50 values from chemical descriptors. All models were extensively validated using special protocols. Conclusions The novelty of this modeling approach is that it uses the relationships between in vivo and in vitro data only to inform the initial construction of the hierarchical two-step QSAR models. Models resulting from this approach employ chemical descriptors only for external prediction of acute rodent toxicity. PMID:19672406

Predicting A Drug'S Membrane Permeability: Evolution of a Computational Model Validated with in Vitro Permeability Assay Data

DOE PAGES

Carpenter, Timothy S.; McNerney, M. Windy; Be, Nicholas A.; ...

2016-02-16

Membrane permeability is a key property to consider in drug design, especially when the drugs in question need to cross the blood-brain barrier (BBB). A comprehensive in vivo assessment of the BBB permeability of a drug takes considerable time and financial resources. A current, simplified in vitro model to investigate drug permeability is a Parallel Artificial Membrane Permeability Assay (PAMPA) that generally provides higher throughput and initial quantification of a drug's passive permeability. Computational methods can also be used to predict drug permeability. Our methods are highly advantageous as they do not require the synthesis of the desired drug, andmore » can be implemented rapidly using high-performance computing. In this study, we have used umbrella sampling Molecular Dynamics (MD) methods to assess the passive permeability of a range of compounds through a lipid bilayer. Furthermore, the permeability of these compounds was comprehensively quantified using the PAMPA assay to calibrate and validate the MD methodology. And after demonstrating a firm correlation between the two approaches, we then implemented our MD method to quantitatively predict the most permeable potential drug from a series of potential scaffolds. This permeability was then confirmed by the in vitro PAMPA methodology. Therefore, in this work we have illustrated the potential that these computational methods hold as useful tools to help predict a drug's permeability in a faster and more cost-effective manner. Release number: LLNL-ABS-677757.« less

Electrochemical Cathodic Polarization, a Simplified Method That Can Modified and Increase the Biological Activity of Titanium Surfaces: A Systematic Review

PubMed Central

2016-01-01

Background The cathodic polarization seems to be an electrochemical method capable of modifying and coat biomolecules on titanium surfaces, improving the surface activity and promoting better biological responses. Objective The aim of the systematic review is to assess the scientific literature to evaluate the cellular response produced by treatment of titanium surfaces by applying the cathodic polarization technique. Data, Sources, and Selection The literature search was performed in several databases including PubMed, Web of Science, Scopus, Science Direct, Scielo and EBSCO Host, until June 2016, with no limits used. Eligibility criteria were used and quality assessment was performed following slightly modified ARRIVE and SYRCLE guidelines for cellular studies and animal research. Results Thirteen studies accomplished the inclusion criteria and were considered in the review. The quality of reporting studies in animal models was low and for the in vitro studies it was high. The in vitro and in vivo results reported that the use of cathodic polarization promoted hydride surfaces, effective deposition, and adhesion of the coated biomolecules. In the experimental groups that used the electrochemical method, cellular viability, proliferation, adhesion, differentiation, or bone growth were better or comparable with the control groups. Conclusions The use of the cathodic polarization method to modify titanium surfaces seems to be an interesting method that could produce active layers and consequently enhance cellular response, in vitro and in vivo animal model studies. PMID:27441840

Predicting A Drug'S Membrane Permeability: Evolution of a Computational Model Validated with in Vitro Permeability Assay Data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carpenter, Timothy S.; McNerney, M. Windy; Be, Nicholas A.

Membrane permeability is a key property to consider in drug design, especially when the drugs in question need to cross the blood-brain barrier (BBB). A comprehensive in vivo assessment of the BBB permeability of a drug takes considerable time and financial resources. A current, simplified in vitro model to investigate drug permeability is a Parallel Artificial Membrane Permeability Assay (PAMPA) that generally provides higher throughput and initial quantification of a drug's passive permeability. Computational methods can also be used to predict drug permeability. Our methods are highly advantageous as they do not require the synthesis of the desired drug, andmore » can be implemented rapidly using high-performance computing. In this study, we have used umbrella sampling Molecular Dynamics (MD) methods to assess the passive permeability of a range of compounds through a lipid bilayer. Furthermore, the permeability of these compounds was comprehensively quantified using the PAMPA assay to calibrate and validate the MD methodology. And after demonstrating a firm correlation between the two approaches, we then implemented our MD method to quantitatively predict the most permeable potential drug from a series of potential scaffolds. This permeability was then confirmed by the in vitro PAMPA methodology. Therefore, in this work we have illustrated the potential that these computational methods hold as useful tools to help predict a drug's permeability in a faster and more cost-effective manner. Release number: LLNL-ABS-677757.« less

Fostering efficacy and toxicity evaluation of traditional Chinese medicine and natural products: Chick embryo as a high throughput model bridging in vitro and in vivo studies.

PubMed

Wu, Tong; Yu, Gui-Yuan; Xiao, Jia; Yan, Chang; Kurihara, Hiroshi; Li, Yi-Fang; So, Kwok-Fai; He, Rong-Rong

2018-04-19

Efficacy and safety assessments are essential thresholds for drug candidates from preclinical to clinical research. Conventional mammalian in vivo models cannot offer rapid pharmacological and toxicological screening, whereas cell-based or cell-free in vitro systems often lead to inaccurate results because of the lack of physiological environment. Within the avian species, gallus gallus is the first bird to have its genome sequencing. Meantime, chick embryo is an easily operating, relatively transparent and extensively accessible model, whose physiological and pathological alterations can be visualized by egg candler, staining and image technologies. These features facilitate chick embryo as a high-throughput screening platform bridging in vivo and in vitro gaps in the pharmaceutical research. Due to the complicated ingredients and multiple-targets natures of traditional Chinese medicine (TCM), testing the efficacy and safety of TCM by in vitro methods are laborious and inaccurate, while testing in mammalian models consume massive cost and time. As such, the productive living organism chick embryo serves as an ideal biological system for pharmacodynamics studies of TCM. Herein, we comprehensively update recent progresses on the specialty of chick embryo in evaluation of efficacy and toxicity of drugs, with special concerns of TCM. Copyright © 2018 Elsevier Ltd. All rights reserved.

Life-Stage Physiologically-Based Pharmacokinetic (PBPK) ...

EPA Pesticide Factsheets

This presentation discusses methods used to extrapolate from in vitro high-throughput screening (HTS) toxicity data for an endocrine pathway to in vivo for early life stages in humans, and the use of a life stage PBPK model to address rapidly changing physiological parameters. Adverse outcome pathways (AOPs), in this case endocrine disruption during development, provide a biologically-based framework for linking molecular initiating events triggered by chemical exposures to key events leading to adverse outcomes. The application of AOPs to human health risk assessment requires extrapolation of in vitro HTS toxicity data to in vivo exposures (IVIVE) in humans, which can be achieved through the use of a PBPK/PD model. Exposure scenarios for chemicals in the PBPK/PD model will consider both placental and lactational transfer of chemicals, with a focus on age dependent dosimetry during fetal development and after birth for a nursing infant. This talk proposes a universal life-stage computational model that incorporates changing physiological parameters to link environmental exposures to in vitro levels of HTS assays related to a developmental toxicological AOP for vascular disruption. In vitro toxicity endpoints discussed are based on two mechanisms: 1) Fetal vascular disruption, and 2) Neurodevelopmental toxicity induced by altering thyroid hormone levels in neonates via inhibition of thyroperoxidase in the thyroid gland. Application of our Life-stage computati

Evaluation of 309 environmental chemicals using a mouse embryonic stem cell adherent cell differentiation and cytotoxicity assay

EPA Science Inventory

The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing development...

Advancing Predictive Hepatotoxicity at the Intersection of Experimental, in Silico, and Artificial Intelligence Technologies.

PubMed

Fraser, Keith; Bruckner, Dylan M; Dordick, Jonathan S

2018-06-18

Adverse drug reactions, particularly those that result in drug-induced liver injury (DILI), are a major cause of drug failure in clinical trials and drug withdrawals. Hepatotoxicity-mediated drug attrition occurs despite substantial investments of time and money in developing cellular assays, animal models, and computational models to predict its occurrence in humans. Underperformance in predicting hepatotoxicity associated with drugs and drug candidates has been attributed to existing gaps in our understanding of the mechanisms involved in driving hepatic injury after these compounds perfuse and are metabolized by the liver. Herein we assess in vitro, in vivo (animal), and in silico strategies used to develop predictive DILI models. We address the effectiveness of several two- and three-dimensional in vitro cellular methods that are frequently employed in hepatotoxicity screens and how they can be used to predict DILI in humans. We also explore how humanized animal models can recapitulate human drug metabolic profiles and associated liver injury. Finally, we highlight the maturation of computational methods for predicting hepatotoxicity, the untapped potential of artificial intelligence for improving in silico DILI screens, and how knowledge acquired from these predictions can shape the refinement of experimental methods.

Recent developments in skin mimic systems to predict transdermal permeation.

PubMed

Waters, Laura J

2015-01-01

In recent years there has been a drive to create experimental techniques that can facilitate the accurate and precise prediction of transdermal permeation without the use of in vivo studies. This review considers why permeation data is essential, provides a brief summary as to how skin acts as a natural barrier to permeation and discusses why in vivo studies are undesirable. This is followed by an in-depth discussion on the extensive range of alternative methods that have been developed in recent years. All of the major 'skin mimic systems' are considered including: in vitro models using synthetic membranes, mathematical models including quantitative structure-permeability relationships (QSPRs), human skin equivalents and chromatographic based methods. All of these model based systems are ideally trying to achieve the same end-point, namely a reliable in vitro-in vivo correlation, i.e. matching non-in vivo obtained data with that from human clinical trials. It is only by achieving this aim, that any new method of obtaining permeation data can be acknowledged as a potential replacement for animal studies, for the determination of transdermal permeation. In this review, the relevance and potential applicability of the various models systems will also be discussed.

In vitro exposure systems and dosimetry assessment tools for inhaled tobacco products: Workshop proceedings, conclusions and paths forward for in vitro model use.

PubMed

Behrsing, Holger; Hill, Erin; Raabe, Hans; Tice, Raymond; Fitzpatrick, Suzanne; Devlin, Robert; Pinkerton, Kent; Oberdörster, Günter; Wright, Chris; Wieczorek, Roman; Aufderheide, Michaela; Steiner, Sandro; Krebs, Tobias; Asgharian, Bahman; Corley, Richard; Oldham, Michael; Adamson, Jason; Li, Xiang; Rahman, Irfan; Grego, Sonia; Chu, Pei-Hsuan; McCullough, Shaun; Curren, Rodger

2017-07-01

In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference entitled, In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products, to bring together stakeholders representing regulatory agencies, academia and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapour exposure systems, as well as the various approaches and challenges to quantifying the complex exposures in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were: a) Tobacco Smoke and E-Cigarette Aerosols; b) Air-Liquid Interface-In Vitro Exposure Systems; c) Dosimetry Approaches for Particles and Vapours/In Vitro Dosimetry Determinations; and d) Exposure Microenvironment/Physiology of Cells. The 2.5-day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will report on the proceedings, recommendations, and outcome of the April 2016 technical workshop, including paths forward for developing and validating non-animal test methods for tobacco product smoke and next generation tobacco product aerosol/vapour exposures. With the recent FDA publication of the final deeming rule for the governance of tobacco products, there is an unprecedented necessity to evaluate a very large number of tobacco-based products and ingredients. The questionable relevance, high cost, and ethical considerations for the use of in vivo testing methods highlight the necessity of robust in vitro approaches to elucidate tobacco-based exposures and how they may lead to pulmonary diseases that contribute to lung exposure-induced mortality worldwide. 2017 FRAME.

Simultaneous determination and stability studies of linezolid, meropenem and vancomycin in bacterial growth medium by high-performance liquid chromatography.

PubMed

Wicha, Sebastian G; Kloft, Charlotte

2016-08-15

For pharmacokinetic/pharmacodynamic (PK/PD) assessment of antibiotics combinations in in vitro infection models, accurate and precise quantification of drug concentrations in bacterial growth medium is crucial for derivation of valid PK/PD relationships. We aimed to (i) develop a high-performance liquid chromatography (HPLC) assay to simultaneously quantify linezolid (LZD), vancomycin (VAN) and meropenem (MER), as typical components of broad-spectrum antibiotic combination therapy, in bacterial growth medium cation-adjusted Mueller-Hinton broth (CaMHB) and (ii) determine the stability profiles of LZD, VAN and MER under conditions in in vitro infection models. To separate sample matrix components, the final method comprised the pretreatment of 100μL sample with 400μL methanol, the evaporation of supernatant and its reconstitution in water. A low sample volume of 2μL processed sample was injected onto an Accucore C-18 column (2.6μm, 100×2.1mm) coupled to a Dionex Ultimate 3000 HPLC+ system. UV detection at 251, 240 and 302nm allowed quantification limits of 0.5, 2 and 0.5μg/mL for LZD, VAN and MER, respectively. The assay was successfully validated according to the relevant EMA guideline. The rapid method (14min) was successfully applied to quantify significant degradation of LZD, VAN and MER in in vitro infection models: LZD was stable, VAN degraded to 90.6% and MER to 62.9% within 24h compared to t=0 in CaMHB at 37°C, which should be considered when deriving PK/PD relationships in in vitro infection models. Inclusion of further antibiotics into the flexible gradient-based HPLC assay seems promising. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a novel and simple method to evaluate disintegration of rapidly disintegrating tablets.

PubMed

Hoashi, Yohei; Tozuka, Yuichi; Takeuchi, Hirofumi

2013-01-01

The purpose of this study was to develop and test a novel and simple method for evaluating the disintegration time of rapidly disintegrating tablets (RDTs) in vitro, since the conventional disintegration test described in the pharmacopoeia produces poor results due to the difference of its environmental conditions from those of an actual oral cavity. Six RDTs prepared in our laboratory and 5 types of commercial RDTs were used as model formulations. Using our original apparatus, a good correlation was observed between in vivo and in vitro disintegration times by adjusting the height from which the solution was dropped to 8 cm and the weight of the load to 10 or 20 g. Properties of RDTs, such as the pattern of their disintegrating process, can be assessed by verifying the load. These findings confirmed that our proposed method for an in vitro disintegration test apparatus is an excellent one for estimating disintegration time and the disintegration profile of RDTs.

In vitro evaluation of antioxidant activity of Cordia dichotoma (Forst f.) bark

PubMed Central

Nariya, Pankaj B.; Bhalodia, Nayan R.; Shukla, Vinay J.; Acharya, Rabinarayan; Nariya, Mukesh B.

2013-01-01

Cordia dichotoma Forst. f. bark, identified as botanical source of Shleshmataka in Ayurvedic pharmacopoeia. Present investigation was undertaken to evaluate possible antioxidant potential of methanolic and butanol extract of C. dichotoma bark. In vitro antioxidant activity of methanolic and butanol extract was determined by 1,1, diphenyl–2, picrylhydrazyl (DPPH) free radical scavenging assay. The extracts were also evaluated for their phenolic contents and antioxidant activity. Phenolic content was measured using Folin–Ciocalteu reagent and was calculated as Gallic acid equivalents. Antiradical activity of methanolic extract was measured by DPPH assay and was compared to ascorbic acid and ferric reducing power of the extract was evaluated by Oyaizu method. In the present study three in vitro models were used to evaluate antioxidant activity. The first two methods were for direct measurement of radical scavenging activity and remaining one method evaluated the reducing power. The present study revealed that the C. dichotoma bark has significant radical scavenging activity. PMID:24049418

In vitro evaluation of antioxidant activity of Cordia dichotoma (Forst f.) bark.

PubMed

Nariya, Pankaj B; Bhalodia, Nayan R; Shukla, Vinay J; Acharya, Rabinarayan; Nariya, Mukesh B

2013-01-01

Cordia dichotoma Forst. f. bark, identified as botanical source of Shleshmataka in Ayurvedic pharmacopoeia. Present investigation was undertaken to evaluate possible antioxidant potential of methanolic and butanol extract of C. dichotoma bark. In vitro antioxidant activity of methanolic and butanol extract was determined by 1,1, diphenyl-2, picrylhydrazyl (DPPH) free radical scavenging assay. The extracts were also evaluated for their phenolic contents and antioxidant activity. Phenolic content was measured using Folin-Ciocalteu reagent and was calculated as Gallic acid equivalents. Antiradical activity of methanolic extract was measured by DPPH assay and was compared to ascorbic acid and ferric reducing power of the extract was evaluated by Oyaizu method. In the present study three in vitro models were used to evaluate antioxidant activity. The first two methods were for direct measurement of radical scavenging activity and remaining one method evaluated the reducing power. The present study revealed that the C. dichotoma bark has significant radical scavenging activity.

Antioxidant activity, total phenolics and flavonoids contents: Should we ban in vitro screening methods?

PubMed

Granato, Daniel; Shahidi, Fereidoon; Wrolstad, Ronald; Kilmartin, Paul; Melton, Laurence D; Hidalgo, Francisco J; Miyashita, Kazuo; Camp, John van; Alasalvar, Cesarettin; Ismail, Amin B; Elmore, Stephen; Birch, Gordon G; Charalampopoulos, Dimitris; Astley, Sian B; Pegg, Ronald; Zhou, Peng; Finglas, Paul

2018-10-30

As many studies are exploring the association between ingestion of bioactive compounds and decreased risk of non-communicable diseases, the scientific community continues to show considerable interest in these compounds. In addition, as many non-nutrients with putative health benefits are reducing agents, hydrogen donors, singlet oxygen quenchers or metal chelators, measurement of antioxidant activity using in vitro assays has become very popular over recent decades. Measuring concentrations of total phenolics, flavonoids, and other compound (sub)classes using UV/Vis spectrophotometry offers a rapid chemical index, but chromatographic techniques are necessary to establish structure-activity. For bioactive purposes, in vivo models are required or, at the very least, methods that employ distinct mechanisms of action (i.e., single electron transfer, transition metal chelating ability, and hydrogen atom transfer). In this regard, better understanding and application of in vitro screening methods should help design of future research studies on 'bioactive compounds'. Copyright © 2018 Elsevier Ltd. All rights reserved.

Life-Stage Physiologically-Based Pharmacokinetic (PBPK) Model Applications to Screen Environmental Hazards.

EPA Science Inventory

This presentation discusses methods used to extrapolate from in vitro high-throughput screening (HTS) toxicity data for an endocrine pathway to in vivo for early life stages in humans, and the use of a life stage PBPK model to address rapidly changing physiological parameters. A...

Using in vitro/in silico data for consumer safety assessment of feed flavoring additives--A feasibility study using piperine.

PubMed

Thiel, A; Etheve, S; Fabian, E; Leeman, W R; Plautz, J R

2015-10-01

Consumer health risk assessment for feed additives is based on the estimated human exposure to the additive that may occur in livestock edible tissues compared to its hazard. We present an approach using alternative methods for consumer health risk assessment. The aim was to use the fewest possible number of animals to estimate its hazard and human exposure without jeopardizing the safety upon use. As an example we selected the feed flavoring substance piperine and applied in silico modeling for residue estimation, results from literature surveys, and Read-Across to assess metabolism in different species. Results were compared to experimental in vitro metabolism data in rat and chicken, and to quantitative analysis of residues' levels from the in vivo situation in livestock. In silico residue modeling showed to be a worst case: the modeled residual levels were considerably higher than the measured residual levels. The in vitro evaluation of livestock versus rodent metabolism revealed no major differences in metabolism between the species. We successfully performed a consumer health risk assessment without performing additional animal experiments. As shown, the use and combination of different alternative methods supports animal welfare consideration and provides future perspective to reducing the number of animals. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

“Do-it-yourself in vitro vasculature that recapitulates in vivo geometries for investigating endothelial-blood cell interactions”

PubMed Central

Mannino, Robert G.; Myers, David R.; Ahn, Byungwook; Wang, Yichen; Margo Rollins; Gole, Hope; Lin, Angela S.; Guldberg, Robert E.; Giddens, Don P.; Timmins, Lucas H.; Lam, Wilbur A.

2015-01-01

Investigating biophysical cellular interactions in the circulation currently requires choosing between in vivo models, which are difficult to interpret due in part to the hemodynamic and geometric complexities of the vasculature; or in vitro systems, which suffer from non-physiologic assumptions and/or require specialized microfabrication facilities and expertise. To bridge that gap, we developed an in vitro “do-it-yourself” perfusable vasculature model that recapitulates in vivo geometries, such as aneurysms, stenoses, and bifurcations, and supports endothelial cell culture. These inexpensive, disposable devices can be created rapidly (<2 hours) with high precision and repeatability, using standard off-the-shelf laboratory supplies. Using these “endothelialized” systems, we demonstrate that spatial variation in vascular cell adhesion molecule (VCAM-1) expression correlates with the wall shear stress patterns of vascular geometries. We further observe that the presence of endothelial cells in stenoses reduces platelet adhesion but increases sickle cell disease (SCD) red blood cell (RBC) adhesion in bifurcations. Overall, our method enables researchers from all disciplines to study cellular interactions in physiologically relevant, yet simple-to-make, in vitro vasculature models. PMID:26202603

Consumer product in vitro digestion model: Bioaccessibility of contaminants and its application in risk assessment.

PubMed

Brandon, Esther F A; Oomen, Agnes G; Rompelberg, Cathy J M; Versantvoort, Carolien H M; van Engelen, Jacqueline G M; Sips, Adrienne J A M

2006-03-01

This paper describes the applicability of in vitro digestion models as a tool for consumer products in (ad hoc) risk assessment. In current risk assessment, oral bioavailability from a specific product is considered to be equal to bioavailability found in toxicity studies in which contaminants are usually ingested via liquids or food matrices. To become bioavailable, contaminants must first be released from the product during the digestion process (i.e. become bioaccessible). Contaminants in consumer products may be less bioaccessible than contaminants in liquid or food. Therefore, the actual risk after oral exposure could be overestimated. This paper describes the applicability of a simple, reliable, fast and relatively inexpensive in vitro method for determining the bioaccessibility of a contaminant from a consumer product. Different models, representing sucking and/or swallowing were developed. The experimental design of each model can be adjusted to the appropriate exposure scenarios as determined by the risk assessor. Several contaminated consumer products were tested in the various models. Although relevant in vivo data are scare, we succeeded to preliminary validate the model for one case. This case showed good correlation and never underestimated the bioavailability. However, validation check needs to be continued.

Engineering cancer microenvironments for in vitro 3-D tumor models

PubMed Central

Asghar, Waseem; El Assal, Rami; Shafiee, Hadi; Pitteri, Sharon; Paulmurugan, Ramasamy; Demirci, Utkan

2017-01-01

The natural microenvironment of tumors is composed of extracellular matrix (ECM), blood vasculature, and supporting stromal cells. The physical characteristics of ECM as well as the cellular components play a vital role in controlling cancer cell proliferation, apoptosis, metabolism, and differentiation. To mimic the tumor microenvironment outside the human body for drug testing, two-dimensional (2-D) and murine tumor models are routinely used. Although these conventional approaches are employed in preclinical studies, they still present challenges. For example, murine tumor models are expensive and difficult to adopt for routine drug screening. On the other hand, 2-D in vitro models are simple to perform, but they do not recapitulate natural tumor microenvironment, because they do not capture important three-dimensional (3-D) cell–cell, cell–matrix signaling pathways, and multi-cellular heterogeneous components of the tumor microenvironment such as stromal and immune cells. The three-dimensional (3-D) in vitro tumor models aim to closely mimic cancer microenvironments and have emerged as an alternative to routinely used methods for drug screening. Herein, we review recent advances in 3-D tumor model generation and highlight directions for future applications in drug testing. PMID:28458612

AlgiMatrix™ Based 3D Cell Culture System as an In-Vitro Tumor Model for Anticancer Studies

PubMed Central

Godugu, Chandraiah; Patel, Apurva R.; Desai, Utkarsh; Andey, Terrick; Sams, Alexandria; Singh, Mandip

2013-01-01

Background Three-dimensional (3D) in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening. Methods Cancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100–300 µm. Spheroids were grown in two weeks in cultures without compromising the growth characteristics. Different marketed anticancer drugs were screened by incubating them for 24 h at 7, 9 and 11 days in 3D cultures and cytotoxicity was measured by AlamarBlue® assay. Effectiveness of anticancer drug treatments were measured based on spheroid number and size distribution. Evaluation of apoptotic and anti-apoptotic markers was done by immunohistochemistry and RT-PCR. The 3D results were compared with the conventional 2D monolayer cultures. Cellular uptake studies for drug (Doxorubicin) and nanoparticle (NLC) were done using spheroids. Results IC50 values for anticancer drugs were significantly higher in AlgiMatrix™ systems compared to 2D culture models. The cleaved caspase-3 expression was significantly decreased (2.09 and 2.47 folds respectively for 5-Fluorouracil and Camptothecin) in H460 spheroid cultures compared to 2D culture system. The cytotoxicity, spheroid size distribution, immunohistochemistry, RT-PCR and nanoparticle penetration data suggested that in vitro tumor models show higher resistance to anticancer drugs and supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro. Conclusion The results from our studies are useful to develop a high throughput in vitro tumor model to study the effect of various anticancer agents and various molecular pathways affected by the anticancer drugs and formulations. PMID:23349734

Rapidly-disintegrating sublingual tablets of epinephrine: role of non-medicinal ingredients in formulation development.

PubMed

Rachid, Ousama; Rawas-Qalaji, Mutasem; Simons, F Estelle R; Simons, Keith J

2012-11-01

Epinephrine is the drug of choice in the management of anaphylaxis. For first-aid treatment in the community, epinephrine autoinjectors (E-autos) are commonly prescribed, but are underutilized. In our laboratory, we developed a series of first-generation rapidly-disintegrating sublingual tablets (RDSTs) containing 40mg of epinephrine. One RDST had similar bioavailability to epinephrine 0.3mg from an auto-injector, as confirmed in a validated rabbit model, while other formulations containing different non-medicinal ingredients (NMIs) and with similar in vitro characteristics demonstrated much lower bioavailability. Subsequently, we evaluated the effect of changing the grade and proportion of NMIs, specifically mannitol and microcrystalline cellulose (MCC), on the in vitro characteristics of second- and third-generation RDSTs. Weight variation, content uniformity, breaking force, and friability were tested using official USP methods. Novel validated methods that simulate ambient conditions of the sublingual cavity were developed to test disintegration time, wetting time, and dissolution. Using these methods, it was possible to measure the effects of making small changes in NMIs on the in vitro characteristics of the formulations. The RDST formulation that resulted in the best in vitro characteristics contained the optimum proportion of mannitol and a specific ratio of coarse and fine particle grades of MCC. Appropriate comparative testing resulted in the selection of the RDST with the optimum in vitro characteristics. Copyright © 2012 Elsevier B.V. All rights reserved.

Risk-based high-throughput chemical screening and prioritization using exposure models and in vitro bioactivity assays

DOE PAGES

Shin, Hyeong -Moo; Ernstoff, Alexi; Arnot, Jon A.; ...

2015-05-01

We present a risk-based high-throughput screening (HTS) method to identify chemicals for potential health concerns or for which additional information is needed. The method is applied to 180 organic chemicals as a case study. We first obtain information on how the chemical is used and identify relevant use scenarios (e.g., dermal application, indoor emissions). For each chemical and use scenario, exposure models are then used to calculate a chemical intake fraction, or a product intake fraction, accounting for chemical properties and the exposed population. We then combine these intake fractions with use scenario-specific estimates of chemical quantity to calculate dailymore » intake rates (iR; mg/kg/day). These intake rates are compared to oral equivalent doses (OED; mg/kg/day), calculated from a suite of ToxCast in vitro bioactivity assays using in vitro-to-in vivo extrapolation and reverse dosimetry. Bioactivity quotients (BQs) are calculated as iR/OED to obtain estimates of potential impact associated with each relevant use scenario. Of the 180 chemicals considered, 38 had maximum iRs exceeding minimum OEDs (i.e., BQs > 1). For most of these compounds, exposures are associated with direct intake, food/oral contact, or dermal exposure. The method provides high-throughput estimates of exposure and important input for decision makers to identify chemicals of concern for further evaluation with additional information or more refined models.« less

In vitro assessment of eye irritancy using the Reconstructed Human Corneal Epithelial SkinEthic HCE model: application to 435 substances from consumer products industry.

PubMed

Cotovio, José; Grandidier, Marie-Hélène; Lelièvre, Damien; Bremond, Christelle; Amsellem, Carolle; Maloug, Saber; Ovigne, Jean-Marc; Loisel-Joubert, Sophie; Lee, Aline Van Der; Minondo, Anne-Marie; Capallere, Christophe; Bertino, Béatrice; Alépée, Nathalie; Tinois-Tessonneaud, Estelle; de Fraissinette, Anne De Brugerolle; Meunier, Jean-Roch; Leclaire, Jacques

2010-03-01

The 7th amendment of the EU Cosmetics Directive led to the ban of eye irritation testing for cosmetic ingredients in animals, effective from March 11th 2009. Over the last 20years, many efforts have been made to find reliable and relevant alternative methods. The SkinEthic HCE model was used to evaluate the in vitro eye irritancy potential of substances from a cosmetic industry portfolio. An optimized protocol based on a specific 1-h treatment and a 16-h post-treatment incubation period was first assessed on a set of 102 substances. The prediction model (PM) based on a 50% viability cut-off, allowed to draw up two classes (Irritants and Non-Irritants), with good associated sensitivity (86.2%) and specificity (83.5%). To check the robustness of the method, the evaluated set was expanded up to 435 substances. Final performances maintained a high level and were characterized by an overall accuracy value > 82% when using EU or GHS classification rules. Results showed that the SkinEthic HCE test method is a promising in vitro tool for the prediction of eye irritancy. Optimization datasets were shared with the COLIPA Eye Irritation Project Team and ECVAM experts, and reviewed as part of an ongoing progression to enter an ECVAM prospective validation study for eye irritation. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

3D artificial bones for bone repair prepared by computed tomography-guided fused deposition modeling for bone repair.

PubMed

Xu, Ning; Ye, Xiaojian; Wei, Daixu; Zhong, Jian; Chen, Yuyun; Xu, Guohua; He, Dannong

2014-09-10

The medical community has expressed significant interest in the development of new types of artificial bones that mimic natural bones. In this study, computed tomography (CT)-guided fused deposition modeling (FDM) was employed to fabricate polycaprolactone (PCL)/hydroxyapatite (HA) and PCL 3D artificial bones to mimic natural goat femurs. The in vitro mechanical properties, in vitro cell biocompatibility, and in vivo performance of the artificial bones in a long load-bearing goat femur bone segmental defect model were studied. All of the results indicate that CT-guided FDM is a simple, convenient, relatively low-cost method that is suitable for fabricating natural bonelike artificial bones. Moreover, PCL/HA 3D artificial bones prepared by CT-guided FDM have more close mechanics to natural bone, good in vitro cell biocompatibility, biodegradation ability, and appropriate in vivo new bone formation ability. Therefore, PCL/HA 3D artificial bones could be potentially be of use in the treatment of patients with clinical bone defects.

Brain Aggregates: An Effective In Vitro Cell Culture System Modeling Neurodegenerative Diseases.

PubMed

Ahn, Misol; Kalume, Franck; Pitstick, Rose; Oehler, Abby; Carlson, George; DeArmond, Stephen J

2016-03-01

Drug discovery for neurodegenerative diseases is particularly challenging because of the discrepancies in drug effects between in vitro and in vivo studies. These discrepancies occur in part because current cell culture systems used for drug screening have many limitations. First, few cell culture systems accurately model human aging or neurodegenerative diseases. Second, drug efficacy may differ between dividing and stationary cells, the latter resembling nondividing neurons in the CNS. Brain aggregates (BrnAggs) derived from embryonic day 15 gestation mouse embryos may represent neuropathogenic processes in prion disease and reflect in vivo drug efficacy. Here, we report a new method for the production of BrnAggs suitable for drug screening and suggest that BrnAggs can model additional neurological diseases such as tauopathies. We also report a functional assay with BrnAggs by measuring electrophysiological activities. Our data suggest that BrnAggs could serve as an effective in vitro cell culture system for drug discovery for neurodegenerative diseases. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

Population dynamics and in vitro antibody pressure of porcine parvovirus indicate a decrease in variability.

PubMed

Streck, André Felipe; Homeier, Timo; Foerster, Tessa; Truyen, Uwe

2013-09-01

To estimate the impact of porcine parvovirus (PPV) vaccines on the emergence of new phenotypes, the population dynamic history of the virus was calculated using the Bayesian Markov chain Monte Carlo method with a Bayesian skyline coalescent model. Additionally, an in vitro model was performed with consecutive passages of the 'Challenge' strain (a virulent field strain) and NADL2 strain (a vaccine strain) in a PK-15 cell line supplemented with polyclonal antibodies raised against the vaccine strain. A decrease in genetic diversity was observed in the presence of antibodies in vitro or after vaccination (as estimated by the in silico model). We hypothesized that the antibodies induced a selective pressure that may reduce the incidence of neutral selection, which should play a major role in the emergence of new mutations. In this scenario, vaccine failures and non-vaccinated populations (e.g. wild boars) may have an important impact in the emergence of new phenotypes.

In Vitro and in Silico Tools To Assess Extent of Cellular Uptake and Lysosomal Sequestration of Respiratory Drugs in Human Alveolar Macrophages.

PubMed

Ufuk, Ayşe; Assmus, Frauke; Francis, Laura; Plumb, Jonathan; Damian, Valeriu; Gertz, Michael; Houston, J Brian; Galetin, Aleksandra

2017-04-03

Accumulation of respiratory drugs in human alveolar macrophages (AMs) has not been extensively studied in vitro and in silico despite its potential impact on therapeutic efficacy and/or occurrence of phospholipidosis. The current study aims to characterize the accumulation and subcellular distribution of drugs with respiratory indication in human AMs and to develop an in silico mechanistic AM model to predict lysosomal accumulation of investigated drugs. The data set included 9 drugs previously investigated in rat AM cell line NR8383. Cell-to-unbound medium concentration ratio (K p,cell ) of all drugs (5 μM) was determined to assess the magnitude of intracellular accumulation. The extent of lysosomal sequestration in freshly isolated human AMs from multiple donors (n = 5) was investigated for clarithromycin and imipramine (positive control) using an indirect in vitro method (±20 mM ammonium chloride, NH 4 Cl). The AM cell parameters and drug physicochemical data were collated to develop an in silico mechanistic AM model. Three in silico models differing in their description of drug membrane partitioning were evaluated; model (1) relied on octanol-water partitioning of drugs, model (2) used in vitro data to account for this process, and model (3) predicted membrane partitioning by incorporating AM phospholipid fractions. In vitro K p,cell ranged >200-fold for respiratory drugs, with the highest accumulation seen for clarithromycin. A good agreement in K p,cell was observed between human AMs and NR8383 (2.45-fold bias), highlighting NR8383 as a potentially useful in vitro surrogate tool to characterize drug accumulation in AMs. The mean K p,cell of clarithromycin (81, CV = 51%) and imipramine (963, CV = 54%) were reduced in the presence of NH 4 Cl by up to 67% and 81%, respectively, suggesting substantial contribution of lysosomal sequestration and intracellular binding in the accumulation of these drugs in human AMs. The in vitro data showed variability in drug accumulation between individual human AM donors due to possible differences in lysosomal abundance, volume, and phospholipid content, which may have important clinical implications. Consideration of drug-acidic phospholipid interactions significantly improved the performance of the in silico models; use of in vitro K p,cell obtained in the presence of NH 4 Cl as a surrogate for membrane partitioning (model (2)) captured the variability in clarithromycin and imipramine K p,cell observed in vitro and showed the best ability to predict correctly positive and negative lysosomotropic properties. The developed mechanistic AM model represents a useful in silico tool to predict lysosomal and cellular drug concentrations based on drug physicochemical data and system specific properties, with potential application to other cell types.

Efficient Genome Editing in Induced Pluripotent Stem Cells with Engineered Nucleases In Vitro.

PubMed

Termglinchan, Vittavat; Seeger, Timon; Chen, Caressa; Wu, Joseph C; Karakikes, Ioannis

2017-01-01

Precision genome engineering is rapidly advancing the application of the induced pluripotent stem cells (iPSCs) technology for in vitro disease modeling of cardiovascular diseases. Targeted genome editing using engineered nucleases is a powerful tool that allows for reverse genetics, genome engineering, and targeted transgene integration experiments to be performed in a precise and predictable manner. However, nuclease-mediated homologous recombination is an inefficient process. Herein, we describe the development of an optimized method combining site-specific nucleases and the piggyBac transposon system for "seamless" genome editing in pluripotent stem cells with high efficiency and fidelity in vitro.

Predicting skin sensitisation using a decision tree integrated testing strategy with an in silico model and in chemico/in vitro assays.

PubMed

Macmillan, Donna S; Canipa, Steven J; Chilton, Martyn L; Williams, Richard V; Barber, Christopher G

2016-04-01

There is a pressing need for non-animal methods to predict skin sensitisation potential and a number of in chemico and in vitro assays have been designed with this in mind. However, some compounds can fall outside the applicability domain of these in chemico/in vitro assays and may not be predicted accurately. Rule-based in silico models such as Derek Nexus are expert-derived from animal and/or human data and the mechanism-based alert domain can take a number of factors into account (e.g. abiotic/biotic activation). Therefore, Derek Nexus may be able to predict for compounds outside the applicability domain of in chemico/in vitro assays. To this end, an integrated testing strategy (ITS) decision tree using Derek Nexus and a maximum of two assays (from DPRA, KeratinoSens, LuSens, h-CLAT and U-SENS) was developed. Generally, the decision tree improved upon other ITS evaluated in this study with positive and negative predictivity calculated as 86% and 81%, respectively. Our results demonstrate that an ITS using an in silico model such as Derek Nexus with a maximum of two in chemico/in vitro assays can predict the sensitising potential of a number of chemicals, including those outside the applicability domain of existing non-animal assays. Copyright © 2016 Elsevier Inc. All rights reserved.

Development of an In Vivo and In Vitro Ileal Fermentation Method in a Growing Pig Model.

PubMed

Montoya, Carlos A; de Haas, Edward S; Moughan, Paul J

2018-02-01

Substantial microbial fermentation may occur mainly in the lower small intestine (SI) of human adults, but there is no established methodology to determine this. The study aimed to develop a combined in vivo and in vitro methodology for ileal fermentation based on the pig as an animal model for digestion in human adults. Several aspects of a combined in vivo/in vitro ileal fermentation assay were evaluated. Male 9-wk-old pigs (n = 30; mean ± SD body weight: 23 ± 1.6 kg) were fed a human-type diet (143, 508, 45, 49, and 116 g/kg dry matter diet of crude protein, starch, total lipid, ash, and total dietary fiber) for 15 d. On day 15, pigs were killed, and the last third of the SI was collected to prepare an ileal digesta-based inoculum. Terminal jejunal digesta (last 50 cm of the second third of the SI) were collected as substrate for the assay to test the form of substrate (fresh or freeze-dried), origin (location in jejunum or SI) of the substrate, storage of the inoculum, incubation time (1.2-6.8 h), pH of the medium, and inoculum concentration (6-26 mg inoculum/100 mg substrate). The group of donor pigs used to prepare the inoculum, form of the substrate, origin of the substrate, origin of the inoculum (location in the SI), storage of the inoculum, incubation time, and inoculum concentration did not influence the in vitro ileal organic matter (OM) fermentability (P > 0.05). The in vitro ileal OM fermentability decreased when the pH of the medium increased from 5.5 to 7.5 (31% to 28%; P ≤ 0.05). Predicted (in vivo/in vitro) apparent ileal OM digestibility was similar to the value measured in vivo. Thirty-percent of the terminal jejunal digesta OM was fermented in the ileum. Fiber fermentation in the ileum can be studied using the optimized in vivo/in vitro ileal fermentation method.

Method to improve passive fit of frameworks on implant-supported prostheses: An in vitro study.

PubMed

Manzella, Carlo; Bignardi, Cristina; Burello, Valerio; Carossa, Stefano; Schierano, Gianmario

2016-07-01

The passivity of the superstructure to the abutments of implant-supported prostheses is necessary for implant-prosthesis success. Improvements are needed in the methods of verifying passivity. The purpose of this in vitro study was to evaluate an inexpensive, easy to make, and user-friendly device to verify the position of the implant abutment replicas of the definitive cast and to avoid framework misfit before fabrication. Eighty stone devices were constructed on a metal base for the in vitro tests. The horizontal, vertical, and angled positions of the implant replicas were created to simulate misfits. The devices were fitted on the abutment replicas, and their ability to identify misfits was evaluated. A statistical analysis was not indicated, because the probability of fracture of the stone devices was 0 or 1. Two mathematical models were built using computer-aided design software (SolidWorks Premium; Dassault Systèmes SolidWorks Corp), and the finite element method was used (Ansys; ANSYS Inc) to simulate the structural behavior of 2 implant configurations (4 and 6 implants). Horizontal misfits of 150 μm, vertical misfits of 50 μm, and angled misfits of 1 degree were detected during the in vitro tests. Different loads and bone quality in the mathematical models did not change stress in the prosthesis configurations on 4 or 6 implants in a relevant way. The fabricated device was easily able to detect the misfits in accordance with the defined parameters. Copyright © 2016 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.

Modeling breath-enhanced jet nebulizers to estimate pulmonary drug deposition.

PubMed

Wee, Wallace B; Leung, Kitty; Coates, Allan L

2013-12-01

Predictable delivery of aerosol medication for a given patient and drug-device combination is crucial, both for therapeutic effect and to avoid toxicity. The gold standard for measuring pulmonary drug deposition (PDD) is gamma scintigraphy. However, these techniques expose patients to radiation, are complicated, and are relevant for only one patient and drug-device combination, making them less available. Alternatively, in vitro experiments have been used as a surrogate to estimate in vivo performance, but this is time-consuming and has few "in vitro to in vivo" correlations for therapeutics delivered by inhalation. An alternative method for determining inhaled mass and PDD is proposed by deriving and validating a mathematical model, for the individual breathing patterns of normal subjects and drug-device operating parameters. This model was evaluated for patients with cystic fibrosis (CF). This study is comprised of three stages: mathematical model derivation, in vitro testing, and in vivo validation. The model was derived from an idealized patient's respiration cycle and the steady-state operating characteristics of a drug-device combination. The model was tested under in vitro dynamic conditions that varied tidal volume, inspiration-to-expiration time, and breaths per minute. This approach was then extended to incorporate additional physiological parameters (dead space, aerodynamic particle size distribution) and validated against in vivo nuclear medicine data in predicting PDD in both normal subjects and those with CF. The model shows strong agreement with in vitro testing. In vivo testing with normal subjects yielded good agreement, but less agreement for patients with chronic obstructive lung disease and bronchiectasis from CF. The mathematical model was successful in accommodating a wide range of breathing patterns and drug-device combinations. Furthermore, the model has demonstrated its effectiveness in predicting the amount of aerosol delivered to "normal" subjects. However, challenges remain in predicting deposition in obstructive lung disease.

Deducing the Kinetics of Protein Synthesis In Vivo from the Transition Rates Measured In Vitro

PubMed Central

Rudorf, Sophia; Thommen, Michael; Rodnina, Marina V.; Lipowsky, Reinhard

2014-01-01

The molecular machinery of life relies on complex multistep processes that involve numerous individual transitions, such as molecular association and dissociation steps, chemical reactions, and mechanical movements. The corresponding transition rates can be typically measured in vitro but not in vivo. Here, we develop a general method to deduce the in-vivo rates from their in-vitro values. The method has two basic components. First, we introduce the kinetic distance, a new concept by which we can quantitatively compare the kinetics of a multistep process in different environments. The kinetic distance depends logarithmically on the transition rates and can be interpreted in terms of the underlying free energy barriers. Second, we minimize the kinetic distance between the in-vitro and the in-vivo process, imposing the constraint that the deduced rates reproduce a known global property such as the overall in-vivo speed. In order to demonstrate the predictive power of our method, we apply it to protein synthesis by ribosomes, a key process of gene expression. We describe the latter process by a codon-specific Markov model with three reaction pathways, corresponding to the initial binding of cognate, near-cognate, and non-cognate tRNA, for which we determine all individual transition rates in vitro. We then predict the in-vivo rates by the constrained minimization procedure and validate these rates by three independent sets of in-vivo data, obtained for codon-dependent translation speeds, codon-specific translation dynamics, and missense error frequencies. In all cases, we find good agreement between theory and experiment without adjusting any fit parameter. The deduced in-vivo rates lead to smaller error frequencies than the known in-vitro rates, primarily by an improved initial selection of tRNA. The method introduced here is relatively simple from a computational point of view and can be applied to any biomolecular process, for which we have detailed information about the in-vitro kinetics. PMID:25358034

Applying fiber optical methods for toxicological testing in vitro

NASA Astrophysics Data System (ADS)

Maerz, Holger K.; Buchholz, Rainer; Emmrich, Frank; Fink, Frank; Geddes, Clive L.; Pfeifer, Lutz; Raabe, Ferdinand; Scheper, Thomas-Helmut; Ulrich, Elizabeth; Marx, Uwe

1999-04-01

The new medical developments, e.g. immune therapy, patient oriented chemotherapy or even gene therapy, create a questionable doubt to the further requirement of animal test. Instead the call for humanitarian reproductive in vitro models becomes increasingly louder. Pharmaceutical usage of in vitro has a long proven history. In cancer research and therapy, the effect of chemostatica in vitro in the so-called oncobiogram is being tested; but the assays do not always correlate with in vivo-like drug resistance and sensitivity. We developed a drug test system in vitro, feasible for therapeutic drug monitoring by the combination of tissue cultivation in hollow fiber bioreactors and fiber optic sensors for monitoring the pharmaceutical effect. Using two fiber optic sensors - an optical oxygen sensor and a metabolism detecting Laserfluoroscope, we were able to successfully monitor the biological status of tissue culture and the drug or toxic effects of in vitro pharmaceutical testing. Furthermore, we developed and patented a system for monitoring the effect of minor toxic compounds which can induce Sick Building Syndrome.

Discovering Synergistic Drug Combination from a Computational Perspective.

PubMed

Ding, Pingjian; Luo, Jiawei; Liang, Cheng; Xiao, Qiu; Cao, Buwen; Li, Guanghui

2018-03-30

Synergistic drug combinations play an important role in the treatment of complex diseases. The identification of effective drug combination is vital to further reduce the side effects and improve therapeutic efficiency. In previous years, in vitro method has been the main route to discover synergistic drug combinations. However, many limitations of time and resource consumption lie within the in vitro method. Therefore, with the rapid development of computational models and the explosive growth of large and phenotypic data, computational methods for discovering synergistic drug combinations are an efficient and promising tool and contribute to precision medicine. It is the key of computational methods how to construct the computational model. Different computational strategies generate different performance. In this review, the recent advancements in computational methods for predicting effective drug combination are concluded from multiple aspects. First, various datasets utilized to discover synergistic drug combinations are summarized. Second, we discussed feature-based approaches and partitioned these methods into two classes including feature-based methods in terms of similarity measure, and feature-based methods in terms of machine learning. Third, we discussed network-based approaches for uncovering synergistic drug combinations. Finally, we analyzed and prospected computational methods for predicting effective drug combinations. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Alginate based 3D hydrogels as an in vitro co-culture model platform for the toxicity screening of new chemical entities.

PubMed

Lan, Shih-Feng; Starly, Binil

2011-10-01

Prediction of human response to potential therapeutic drugs is through conventional methods of in vitro cell culture assays and expensive in vivo animal testing. Alternatives to animal testing require sophisticated in vitro model systems that must replicate in vivo like function for reliable testing applications. Advancements in biomaterials have enabled the development of three-dimensional (3D) cell encapsulated hydrogels as in vitro drug screening tissue model systems. In this study, we have developed an in vitro platform to enable high density 3D culture of liver cells combined with a monolayer growth of target breast cancer cell line (MCF-7) in a static environment as a representative example of screening drug compounds for hepatotoxicity and drug efficacy. Alginate hydrogels encapsulated with serial cell densities of HepG2 cells (10(5)-10(8) cells/ml) are supported by a porous poly-carbonate disc platform and co-cultured with MCF-7 cells within standard cell culture plates during a 3 day study period. The clearance rates of drug transformation by HepG2 cells are measured using a coumarin based pro-drug. The platform was used to test for HepG2 cytotoxicity 50% (CT(50)) using commercially available drugs which further correlated well with published in vivo LD(50) values. The developed test platform allowed us to evaluate drug dose concentrations to predict hepatotoxicity and its effect on the target cells. The in vitro 3D co-culture platform provides a scalable and flexible approach to test multiple-cell types in a hybrid setting within standard cell culture plates which may open up novel 3D in vitro culture techniques to screen new chemical entity compounds. Copyright © 2011 Elsevier Inc. All rights reserved.

Studies on the antioxidant activities of natural vanilla extract and its constituent compounds through in vitro models.

PubMed

Shyamala, B N; Naidu, M Madhava; Sulochanamma, G; Srinivas, P

2007-09-19

Vanilla extract was prepared by extraction of cured vanilla beans with aqueous ethyl alcohol (60%). The extract was profiled by HPLC, wherein major compounds, viz., vanillic acid, 4-hydroxybenzyl alcohol, 4-hydroxy-3-methoxybenzyl alcohol, 4-hydroxybenzaldehyde and vanillin, could be identified and separated. Extract and pure standard compounds were screened for antioxidant activity using beta-carotene-linoleate and DPPH in vitro model systems. At a concentration of 200 ppm, the extract showed 26% and 43% of antioxidant activity by beta-carotene-linoleate and DPPH methods, respectively, in comparison to corresponding values of 93% and 92% for BHA. Interestingly, 4-hydroxy-3-methoxybenzyl alcohol and 4-hydroxybenzyl alcohol exhibited antioxidant activity of 65% and 45% by beta-carotene-linoleate method and 90% and 50% by DPPH methods, respectively. In contrast, pure vanillin exhibited much lower antioxidant activity. The present study points toward the potential use of vanilla extract components as antioxidants for food preservation and in health supplements as nutraceuticals.

In-vitro evaluation of the accuracy of conventional and digital methods of obtaining full-arch dental impressions.

PubMed

Ender, Andreas; Mehl, Albert

2015-01-01

To investigate the accuracy of conventional and digital impression methods used to obtain full-arch impressions by using an in-vitro reference model. Eight different conventional (polyether, POE; vinylsiloxanether, VSE; direct scannable vinylsiloxanether, VSES; and irreversible hydrocolloid, ALG) and digital (CEREC Bluecam, CER; CEREC Omnicam, OC; Cadent iTero, ITE; and Lava COS, LAV) full-arch impressions were obtained from a reference model with a known morphology, using a highly accurate reference scanner. The impressions obtained were then compared with the original geometry of the reference model and within each test group. A point-to-point measurement of the surface of the model using the signed nearest neighbour method resulted in a mean (10%-90%)/2 percentile value for the difference between the impression and original model (trueness) as well as the difference between impressions within a test group (precision). Trueness values ranged from 11.5 μm (VSE) to 60.2 μm (POE), and precision ranged from 12.3 μm (VSE) to 66.7 μm (POE). Among the test groups, VSE, VSES, and CER showed the highest trueness and precision. The deviation pattern varied with the impression method. Conventional impressions showed high accuracy across the full dental arch in all groups, except POE and ALG. Conventional and digital impression methods show differences regarding full-arch accuracy. Digital impression systems reveal higher local deviations of the full-arch model. Digital intraoral impression systems do not show superior accuracy compared to highly accurate conventional impression techniques. However, they provide excellent clinical results within their indications applying the correct scanning technique.

Growing Arabidopsis in vitro: cell suspensions, in vitro culture, and regeneration.

PubMed

Barkla, Bronwyn J; Vera-Estrella, Rosario; Pantoja, Omar

2014-01-01

An understanding of basic methods in Arabidopsis tissue culture is beneficial for any laboratory working on this model plant. Tissue culture refers to the aseptic growth of cells, organs, or plants in a controlled environment, in which physical, nutrient, and hormonal conditions can all be easily manipulated and monitored. The methodology facilitates the production of a large number of plants that are genetically identical over a relatively short growth period. Techniques, including callus production, cell suspension cultures, and plant regeneration, are all indispensable tools for the study of cellular biochemical and molecular processes. Plant regeneration is a key technology for successful stable plant transformation, while cell suspension cultures can be exploited for metabolite profiling and mining. In this chapter we report methods for the successful and highly efficient in vitro regeneration of plants and production of stable cell suspension lines from leaf explants of both Arabidopsis thaliana and Arabidopsis halleri.

Systematic comparison of the behaviors produced by computational models of epileptic neocortex.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warlaumont, A. S.; Lee, H. C.; Benayoun, M.

2010-12-01

Two existing models of brain dynamics in epilepsy, one detailed (i.e., realistic) and one abstract (i.e., simplified) are compared in terms of behavioral range and match to in vitro mouse recordings. A new method is introduced for comparing across computational models that may have very different forms. First, high-level metrics were extracted from model and in vitro output time series. A principal components analysis was then performed over these metrics to obtain a reduced set of derived features. These features define a low-dimensional behavior space in which quantitative measures of behavioral range and degree of match to real data canmore » be obtained. The detailed and abstract models and the mouse recordings overlapped considerably in behavior space. Both the range of behaviors and similarity to mouse data were similar between the detailed and abstract models. When no high-level metrics were used and principal components analysis was computed over raw time series, the models overlapped minimally with the mouse recordings. The method introduced here is suitable for comparing across different kinds of model data and across real brain recordings. It appears that, despite differences in form and computational expense, detailed and abstract models do not necessarily differ in their behaviors.« less

Selection of animal models for pre-clinical strategies in evaluating the fracture healing, bone graft substitutes and bone tissue regeneration and engineering.

PubMed

Bigham-Sadegh, Amin; Oryan, Ahmad

2015-06-01

In vitro assays can be useful in determining biological mechanism and optimizing scaffold parameters, however translation of the in vitro results to clinics is generally hard. Animal experimentation is a better approximation than in vitro tests, and usage of animal models is often essential in extrapolating the experimental results and translating the information in a human clinical setting. In addition, usage of animal models to study fracture healing is useful to answer questions related to the most effective method to treat humans. There are several factors that should be considered when selecting an animal model. These include availability of the animal, cost, ease of handling and care, size of the animal, acceptability to society, resistance to surgery, infection and disease, biological properties analogous to humans, bone structure and composition, as well as bone modeling and remodeling characteristics. Animal experiments on bone healing have been conducted on small and large animals, including mice, rats, rabbits, dogs, pigs, goats and sheep. This review also describes the molecular events during various steps of fracture healing and explains different means of fracture healing evaluation including biomechanical, histopathological and radiological assessments.

Microfluidic neurite guidance to study structure-function relationships in topologically-complex population-based neural networks.

PubMed

Honegger, Thibault; Thielen, Moritz I; Feizi, Soheil; Sanjana, Neville E; Voldman, Joel

2016-06-22

The central nervous system is a dense, layered, 3D interconnected network of populations of neurons, and thus recapitulating that complexity for in vitro CNS models requires methods that can create defined topologically-complex neuronal networks. Several three-dimensional patterning approaches have been developed but none have demonstrated the ability to control the connections between populations of neurons. Here we report a method using AC electrokinetic forces that can guide, accelerate, slow down and push up neurites in un-modified collagen scaffolds. We present a means to create in vitro neural networks of arbitrary complexity by using such forces to create 3D intersections of primary neuronal populations that are plated in a 2D plane. We report for the first time in vitro basic brain motifs that have been previously observed in vivo and show that their functional network is highly decorrelated to their structure. This platform can provide building blocks to reproduce in vitro the complexity of neural circuits and provide a minimalistic environment to study the structure-function relationship of the brain circuitry.

Microfluidic neurite guidance to study structure-function relationships in topologically-complex population-based neural networks

NASA Astrophysics Data System (ADS)

Honegger, Thibault; Thielen, Moritz I.; Feizi, Soheil; Sanjana, Neville E.; Voldman, Joel

2016-06-01

The central nervous system is a dense, layered, 3D interconnected network of populations of neurons, and thus recapitulating that complexity for in vitro CNS models requires methods that can create defined topologically-complex neuronal networks. Several three-dimensional patterning approaches have been developed but none have demonstrated the ability to control the connections between populations of neurons. Here we report a method using AC electrokinetic forces that can guide, accelerate, slow down and push up neurites in un-modified collagen scaffolds. We present a means to create in vitro neural networks of arbitrary complexity by using such forces to create 3D intersections of primary neuronal populations that are plated in a 2D plane. We report for the first time in vitro basic brain motifs that have been previously observed in vivo and show that their functional network is highly decorrelated to their structure. This platform can provide building blocks to reproduce in vitro the complexity of neural circuits and provide a minimalistic environment to study the structure-function relationship of the brain circuitry.

An ex vivo approach to botanical-drug interactions: A proof of concept study

PubMed Central

Wang, Xinwen; Zhu, Hao-Jie; Munoz, Juliana; Gurley, Bill J.; Markowitz, John S.

2015-01-01

Ethnopharmacological relevance Botanical medicines are frequently used in combination with therapeutic drugs, imposing a risk for harmful botanical-drug interactions (BDIs). Among the existing BDI evaluation methods, clinical studies are the most desirable, but due to their expense and protracted time-line for completion, conventional in vitro methodologies remain the most frequently used BDI assessment tools. However, many predictions generated from in vitro studies are inconsistent with clinical findings. Accordingly, the present study aimed to develop a novel ex vivo approach for BDI assessment and expand the safety evaluation methodoloy in applied ethnopharmacological research. Materials and Methods This approach differs from conventional in vitro methods in that rather than botanical extracts or individual phytochemicals being prepared in artificial buffers, human plasma/serum collected from a limited number of subjects administered botanical supplements was utilized to assess BDIs. To validate the methodology, human plasma/serum samples collected from healthy subjects administered either milk thistle or goldenseal extracts were utilized in incubation studies to determine their potential inhibitory effects on CYP2C9 and CYP3A4/5, respectively. Silybin A and B, two principal milk thistle phytochemicals, and hydrastine and berberine, the purported active constituents in goldenseal, were evaluated in both phosphate buffer and human plasma based in vitro incubation systems. Results Ex vivo study results were consistent with formal clinical study findings for the effect of milk thistle on the disposition of tolbutamide, a CYP2C9 substrate, and for goldenseal’s influence on the pharmacokinetics of midazolam, a widely accepted CYP3A4/5 substrate. Compared to conventional in vitro BDI methodologies of assessment, the introduction of human plasma into the in vitro study model changed the observed inhibitory effect of silybinA, silybin B and hydrastine and berberine on CYP2C9 and CYP3A4/5, respectively, results which more closely mirrored those generated in clinical study. Conclusions Data from conventional buffer-based in vitro studies were less predictive than the ex vivo assessments. Thus, this novel ex vivo approach may be more effective at predicting clinically relevant BDIs than conventional in vitro methods. PMID:25623616

Generalized in vitro-in vivo relationship (IVIVR) model based on artificial neural networks

PubMed Central

Mendyk, Aleksander; Tuszyński, Paweł K; Polak, Sebastian; Jachowicz, Renata

2013-01-01

Background The aim of this study was to develop a generalized in vitro-in vivo relationship (IVIVR) model based on in vitro dissolution profiles together with quantitative and qualitative composition of dosage formulations as covariates. Such a model would be of substantial aid in the early stages of development of a pharmaceutical formulation, when no in vivo results are yet available and it is impossible to create a classical in vitro-in vivo correlation (IVIVC)/IVIVR. Methods Chemoinformatics software was used to compute the molecular descriptors of drug substances (ie, active pharmaceutical ingredients) and excipients. The data were collected from the literature. Artificial neural networks were used as the modeling tool. The training process was carried out using the 10-fold cross-validation technique. Results The database contained 93 formulations with 307 inputs initially, and was later limited to 28 in a course of sensitivity analysis. The four best models were introduced into the artificial neural network ensemble. Complete in vivo profiles were predicted accurately for 37.6% of the formulations. Conclusion It has been shown that artificial neural networks can be an effective predictive tool for constructing IVIVR in an integrated generalized model for various formulations. Because IVIVC/IVIVR is classically conducted for 2–4 formulations and with a single active pharmaceutical ingredient, the approach described here is unique in that it incorporates various active pharmaceutical ingredients and dosage forms into a single model. Thus, preliminary IVIVC/IVIVR can be available without in vivo data, which is impossible using current IVIVC/IVIVR procedures. PMID:23569360

Plasma-deposited tetraglyme surfaces greatly reduce total blood protein adsorption, contact activation, platelet adhesion, platelet procoagulant activity, and in vitro thrombus deposition.

PubMed

Cao, Lan; Chang, Mark; Lee, Chi-Ying; Castner, David G; Sukavaneshvar, Sivaprasad; Ratner, Buddy D; Horbett, Thomas A

2007-06-15

The ability of tetraethylene glycol dimethyl ether (tetraglyme) plasma deposited coatings exhibiting ultralow fibrinogen adsorption to reduce blood activation was studied with six in vitro methods, namely fibrinogen and von Willebrand's factor adsorption, total protein adsorption, clotting time in recalcified plasma, platelet adhesion and procoagulant activity, and whole blood thrombosis in a disturbed flow catheter model. Surface plasmon resonance results showed that tetraglyme surfaces strongly resisted the adsorption of all proteins from human plasma. The clotting time in the presence of tetraglyme surfaces was lengthened compared with controls, indicating a lower activation of the intrinsic coagulation cascade. Platelet adhesion and thrombin generation by adherent platelets were greatly reduced on tetraglyme-coated materials, compared with uncoated and Biospan-coated glass slides. In the in vitro disturbed blood flow model, tetraglyme plasma coated catheters had 50% less thrombus than did the uncoated catheters. Tetraglyme-coated materials thus had greatly reduced blood interactions as measured with all six methods. The improved blood compatibility of plasma-deposited tetraglyme is thus not only due to their reduced platelet adhesion and activation, but also to a generalized reduction in blood interactions. (c) 2007 Wiley Periodicals, Inc.

Development and Application of Computational/In Vitro Toxicological Methods for Chemical Hazard Risk Reduction of New Materials for Advanced Weapon Systems

NASA Technical Reports Server (NTRS)

Frazier, John M.; Mattie, D. R.; Hussain, Saber; Pachter, Ruth; Boatz, Jerry; Hawkins, T. W.

2000-01-01

The development of quantitative structure-activity relationship (QSAR) is essential for reducing the chemical hazards of new weapon systems. The current collaboration between HEST (toxicology research and testing), MLPJ (computational chemistry) and PRS (computational chemistry, new propellant synthesis) is focusing R&D efforts on basic research goals that will rapidly transition to useful products for propellant development. Computational methods are being investigated that will assist in forecasting cellular toxicological end-points. Models developed from these chemical structure-toxicity relationships are useful for the prediction of the toxicological endpoints of new related compounds. Research is focusing on the evaluation tools to be used for the discovery of such relationships and the development of models of the mechanisms of action. Combinations of computational chemistry techniques, in vitro toxicity methods, and statistical correlations, will be employed to develop and explore potential predictive relationships; results for series of molecular systems that demonstrate the viability of this approach are reported. A number of hydrazine salts have been synthesized for evaluation. Computational chemistry methods are being used to elucidate the mechanism of action of these salts. Toxicity endpoints such as viability (LDH) and changes in enzyme activity (glutahoione peroxidase and catalase) are being experimentally measured as indicators of cellular damage. Extrapolation from computational/in vitro studies to human toxicity, is the ultimate goal. The product of this program will be a predictive tool to assist in the development of new, less toxic propellants.

Antibacterial Effects of Toothpastes Evaluated in an 
In Vitro Biofilm Model.

PubMed

Fernández, Eva; Sánchez, María Del Carmen; Llama-Palacios, Arancha; Sanz, Mariano; Herrera, David

To test the antibacterial effects of different toothpastes with the slurry method of toothpaste application in an in vitro oral biofilm model including relevant periodontal pathogens. Four commercially available toothpastes, two containing sodium fluoride (NaF) at different concentrations (1450 and 2500 ppm), two NaF with either triclosan or stannous fluoride, and a control phosphate-buffered saline (PBS) were used. Multispecies biofilms containing 6 species of oral bacteria were grown on hydroxyapatite disks for 72 h and then exposed for 2 min to the toothpaste slurries or phosphate buffer saline (PBS) by immersion, under continuous agitation at 37°C. Biofilms were then analysed by means of real-time polymerase chain reaction (PCR), combined with propidium monoazide (PMA). Statistical evaluation was performed using ANOVA and Student's t-test, with Bonferroni correction for multiple comparisons. The toothpastes containing NaF and stannous fluoride demonstrated superior antimicrobial activity for A. actinomycetencomitans, P. gingivalis and F. nucleatum when compared to those containing NaF and triclosan, 1450 ppm NaF or 2500 ppm NaF in this multispecies biofilm model. The proposed model for the evaluation of toothpastes in the form of slurries detected significant differences in the antimicrobial effects among the tested NaF-containing toothpastes, with the stannous fluoride-based formulation achieving better results than the other formulations. The use of toothpaste as slurries and real-time PCR with PMA is an adequate method for comparing the in vitro antimicrobial effect of different toothpastes.

Development of a Physiologically Based Pharmacokinetic Model for Sinogliatin, a First-in-Class Glucokinase Activator, by Integrating Allometric Scaling, In Vitro to In Vivo Exploration and Steady-State Concentration-Mean Residence Time Methods: Mechanistic Understanding of its Pharmacokinetics.

PubMed

Song, Ling; Zhang, Yi; Jiang, Ji; Ren, Shuang; Chen, Li; Liu, Dongyang; Chen, Xijing; Hu, Pei

2018-04-06

The objective of this study was to develop a physiologically based pharmacokinetic (PBPK) model for sinogliatin (HMS-5552, dorzagliatin) by integrating allometric scaling (AS), in vitro to in vivo exploration (IVIVE), and steady-state concentration-mean residence time (C ss -MRT) methods and to provide mechanistic insight into its pharmacokinetic properties in humans. Human major pharmacokinetic parameters were analyzed using AS, IVIVE, and C ss -MRT methods with available preclinical in vitro and in vivo data to understand sinogliatin drug metabolism and pharmacokinetic (DMPK) characteristics and underlying mechanisms. On this basis, an initial mechanistic PBPK model of sinogliatin was developed. The initial PBPK model was verified using observed data from a single ascending dose (SAD) study and further optimized with various strategies. The final model was validated by simulating sinogliatin pharmacokinetics under a fed condition. The validated model was applied to support a clinical drug-drug interaction (DDI) study design and to evaluate the effects of intrinsic (hepatic cirrhosis, genetic) factors on drug exposure. The two-species scaling method using rat and dog data (TS- rat,dog ) was the best AS method in predicting human systemic clearance in the central compartment (CL). The IVIVE method confirmed that sinogliatin was predominantly metabolized by cytochrome P450 (CYP) 3A4. The C ss -MRT method suggested dog pharmacokinetic profiles were more similar to human pharmacokinetic profiles. The estimated CL using the AS and IVIVE approaches was within 1.5-fold of that observed. The C ss -MRT method in dogs also provided acceptable prediction of human pharmacokinetic characteristics. For the PBPK approach, the 90% confidence intervals (CIs) of the simulated maximum concentration (C max ), CL, and area under the plasma concentration-time curve (AUC) of sinogliatin were within those observed and the 90% CI of simulated time to C max (t max ) was closed to that observed for a dose range of 5-50 mg in the SAD study. The final PBPK model was validated by simulating sinogliatin pharmacokinetics with food. The 90% CIs of the simulated C max , CL, and AUC values for sinogliatin were within those observed and the 90% CI of the simulated t max was partially within that observed for the dose range of 25-200 mg in the multiple ascending dose (MAD) study. This PBPK model selected a final clinical DDI study design with itraconazole from four potential designs and also evaluated the effects of intrinsic (hepatic cirrhosis, genetic) factors on drug exposure. Sinogliatin pharmacokinetic properties were mechanistically understood by integrating all four methods and a mechanistic PBPK model was successfully developed and validated using clinical data. This PBPK model was applied to support the development of sinogliatin.

A finite element method model to simulate laser interstitial thermo therapy in anatomical inhomogeneous regions

PubMed Central

Mohammed, Yassene; Verhey, Janko F

2005-01-01

Background Laser Interstitial ThermoTherapy (LITT) is a well established surgical method. The use of LITT is so far limited to homogeneous tissues, e.g. the liver. One of the reasons is the limited capability of existing treatment planning models to calculate accurately the damage zone. The treatment planning in inhomogeneous tissues, especially of regions near main vessels, poses still a challenge. In order to extend the application of LITT to a wider range of anatomical regions new simulation methods are needed. The model described with this article enables efficient simulation for predicting damaged tissue as a basis for a future laser-surgical planning system. Previously we described the dependency of the model on geometry. With the presented paper including two video files we focus on the methodological, physical and mathematical background of the model. Methods In contrast to previous simulation attempts, our model is based on finite element method (FEM). We propose the use of LITT, in sensitive areas such as the neck region to treat tumours in lymph node with dimensions of 0.5 cm – 2 cm in diameter near the carotid artery. Our model is based on calculations describing the light distribution using the diffusion approximation of the transport theory; the temperature rise using the bioheat equation, including the effect of microperfusion in tissue to determine the extent of thermal damage; and the dependency of thermal and optical properties on the temperature and the injury. Injury is estimated using a damage integral. To check our model we performed a first in vitro experiment on porcine muscle tissue. Results We performed the derivation of the geometry from 3D ultrasound data and show for this proposed geometry the energy distribution, the heat elevation, and the damage zone. Further on, we perform a comparison with the in-vitro experiment. The calculation shows an error of 5% in the x-axis parallel to the blood vessel. Conclusions The FEM technique proposed can overcome limitations of other methods and enables an efficient simulation for predicting the damage zone induced using LITT. Our calculations show clearly that major vessels would not be damaged. The area/volume of the damaged zone calculated from both simulation and in-vitro experiment fits well and the deviation is small. One of the main reasons for the deviation is the lack of accurate values of the tissue optical properties. In further experiments this needs to be validated. PMID:15631630

Blood-brain barrier structure and function and the challenges for CNS drug delivery.

PubMed

Abbott, N Joan

2013-05-01

The neurons of the central nervous system (CNS) require precise control of their bathing microenvironment for optimal function, and an important element in this control is the blood-brain barrier (BBB). The BBB is formed by the endothelial cells lining the brain microvessels, under the inductive influence of neighbouring cell types within the 'neurovascular unit' (NVU) including astrocytes and pericytes. The endothelium forms the major interface between the blood and the CNS, and by a combination of low passive permeability and presence of specific transport systems, enzymes and receptors regulates molecular and cellular traffic across the barrier layer. A number of methods and models are available for examining BBB permeation in vivo and in vitro, and can give valuable information on the mechanisms by which therapeutic agents and constructs permeate, ways to optimize permeation, and implications for drug discovery, delivery and toxicity. For treating lysosomal storage diseases (LSDs), models can be included that mimic aspects of the disease, including genetically-modified animals, and in vitro models can be used to examine the effects of cells of the NVU on the BBB under pathological conditions. For testing CNS drug delivery, several in vitro models now provide reliable prediction of penetration of drugs including large molecules and artificial constructs with promising potential in treating LSDs. For many of these diseases it is still not clear how best to deliver appropriate drugs to the CNS, and a concerted approach using a variety of models and methods can give critical insights and indicate practical solutions.

In vitro eye irritation testing using the open source reconstructed hemicornea - a ring trial.

PubMed

Mewes, Karsten R; Engelke, Maria; Zorn-Kruppa, Michaela; Bartok, Melinda; Tandon, Rashmi; Brandner, Johanna M; Petersohn, Dirk

2017-01-01

The aim of the present ring trial was to test whether two new methodological approaches for the in vitro classification of eye irritating chemicals can be reliably transferred from the developers' laboratories to other sites. Both test methods are based on the well-established open source reconstructed 3D hemicornea models. In the first approach, the initial depth of injury after chemical treatment in the hemicornea model is derived from the quantitative analysis of histological sections. In the second approach, tissue viability, as a measure for corneal damage after chemical treatment, is analyzed separately for epithelium and stroma of the hemicornea model. The three independent laboratories that participated in the ring trial produced their own hemicornea models according to the test producer's instructions, thus supporting the open source concept. A total of 9 chemicals with different physicochemical and eye-irritating properties were tested to assess the between-laboratory reproducibility (BLR), the predictive performance, as well as possible limitations of the test systems. The BLR was 62.5% for the first and 100% for the second method. Both methods enabled to discriminate Cat. 1 chemicals from all non-Cat. 1 substances, which qualifies them to be used in a top-down approach. However, the selectivity between No Cat. and Cat. 2 chemicals still needs optimization.

Analysis of in vitro fertilization data with multiple outcomes using discrete time-to-event analysis

PubMed Central

Maity, Arnab; Williams, Paige; Ryan, Louise; Missmer, Stacey; Coull, Brent; Hauser, Russ

2014-01-01

In vitro fertilization (IVF) is an increasingly common method of assisted reproductive technology. Because of the careful observation and followup required as part of the procedure, IVF studies provide an ideal opportunity to identify and assess clinical and demographic factors along with environmental exposures that may impact successful reproduction. A major challenge in analyzing data from IVF studies is handling the complexity and multiplicity of outcome, resulting from both multiple opportunities for pregnancy loss within a single IVF cycle in addition to multiple IVF cycles. To date, most evaluations of IVF studies do not make use of full data due to its complex structure. In this paper, we develop statistical methodology for analysis of IVF data with multiple cycles and possibly multiple failure types observed for each individual. We develop a general analysis framework based on a generalized linear modeling formulation that allows implementation of various types of models including shared frailty models, failure specific frailty models, and transitional models, using standard software. We apply our methodology to data from an IVF study conducted at the Brigham and Women’s Hospital, Massachusetts. We also summarize the performance of our proposed methods based on a simulation study. PMID:24317880

Impact of an Anticaries Mouthrinse on In Vitro Remineralization and Microbial Control

PubMed Central

Sun, Frank C.; Engelman, E. Eric; McGuire, James A.; Kosmoski, Gabrielle; Carratello, Lauren; Ricci-Nittel, Danette; Zhang, Jane Z.; Schemehorn, Bruce R.; Gambogi, Robert J.

2014-01-01

Objective. The objective of this research was to evaluate the caries control potential of a new fluoride mouthrinse that also contained antimicrobial agents and a biofilm disrupting agent using different in vitro models. Methods. Four in vitro studies were conducted to assess the performance of this three pronged approach to caries control: (1) traditional enamel fluoride uptake, (2) surface microhardness study using pH cycling model and subsequent fluoride uptake, (3) a salivary biofilm flow-through study to determine the anti-microbial activity, and (4) a single species biofilm model measuring effect on biofilm matrix disruption. Results. The data showed that a LISTERINE rinse with fluoride, essential oils and xylitol was superior in promoting enamel fluoride uptake and in enhancing antimicrobial activity over traditional commercially available fluoridated products. An increase of the surface microhardness was observed when the LISTERINE rinse was used in combination with fluoridated toothpaste versus the fluoridated toothpaste alone. Finally, it was demonstrated that xylitol solutions disrupted and reduced the biovolume of biofilm matrix of mature Streptococcus mutans. Conclusion. These in vitro studies demonstrated that a fluoride mouthrinse with antimicrobial agent and biofilm matrix disrupting agent provided multifaceted and enhanced anti-caries efficacy by promoting remineralization, reducing acidogenic bacteria and disrupting biofilm matrix. PMID:24648842

Antinociceptive and Antioxidant Activities of Phytol In Vivo and In Vitro Models

PubMed Central

Santos, Camila Carolina de Menezes Patrício; Salvadori, Mirian Stiebbe; Mota, Vanine Gomes; Costa, Luciana Muratori; de Almeida, Antonia Amanda Cardoso; de Oliveira, Guilherme Antônio Lopes; Costa, Jéssica Pereira; de Freitas, Rivelilson Mendes; de Almeida, Reinaldo Nóbrega

2013-01-01

The objective of the present study was to evaluate the antinociceptive effects of phytol using chemical and thermal models of nociception in mice and to assess its antioxidant effects in vitro. Phytol was administered intraperitoneally (i.p.) to mice at doses of 25, 50, 100, and 200 mg/kg. In the acetic acid-induced writhing test, phytol significantly reduced the number of contortions compared to the control group (P < 0.001). In the formalin test, phytol reduced significantly the amount of time spent in paw licking in both phases (the neurogenic and inflammatory phases), this effect being more pronounced in the second phase (P < 0.001). Phytol also provoked a significant increase in latency in the hot plate test. These antinociceptive effects did not impaire the motor performance, as shown in the rotarod test. Phytol demonstrated a strong antioxidant effect in vitro in its capacity to remove hydroxyl radicals and nitric oxide as well as to prevent the formation of thiobarbituric acid reactive substances (TBARS). Taken as a whole, these results show the pronounced antinociceptive effects of phytol in the nociception models used, both through its central and peripheral actions, but also its antioxidant properties demonstrated in the in vitro methods used. PMID:26317107

In Vitro versus In Vivo Phase Instability of Zirconia-Toughened Alumina Femoral Heads: A Critical Comparative Assessment

PubMed Central

Pezzotti, Giuseppe; Affatato, Saverio; Rondinella, Alfredo; Yorifuji, Makiko; Marin, Elia; Zhu, Wenliang; McEntire, Bryan; Bal, Sonny B.; Yamamoto, Kengo

2017-01-01

A clear discrepancy between predicted in vitro and actual in vivo surface phase stability of BIOLOX®delta zirconia-toughened alumina (ZTA) femoral heads has been demonstrated by several independent research groups. Data from retrievals challenge the validity of the standard method currently utilized in evaluating surface stability and raise a series of important questions: (1) Why do in vitro hydrothermal aging treatments conspicuously fail to model actual results from the in vivo environment? (2) What is the preponderant microscopic phenomenon triggering the accelerated transformation in vivo? (3) Ultimately, what revisions of the current in vitro standard are needed in order to obtain consistent predictions of ZTA transformation kinetics in vivo? Reported in this paper is a new in toto method for visualizing the surface stability of femoral heads. It is based on CAD-assisted Raman spectroscopy to quantitatively assess the phase transformation observed in ZTA retrievals. Using a series of independent analytical probes, an evaluation of the microscopic mechanisms responsible for the polymorphic transformation is also provided. An outline is given of the possible ways in which the current hydrothermal simulation standard for artificial joints can be improved in an attempt to reduce the gap between in vitro simulation and reality. PMID:28772828

Purification of SUMO conjugating enzymes and kinetic analysis of substrate conjugation

PubMed Central

Yunus, Ali A.; Lima, Christopher D.

2009-01-01

SUMO conjugation to protein substrates requires the concerted action of a dedicated E2 ubiquitin conjugation enzyme (Ubc9) and associated E3 ligases. Although Ubc9 can directly recognize and modify substrate lysine residues that occur within a consensus site for SUMO modification, E3 ligases can redirect specificity and enhance conjugation rates during SUMO conjugation in vitro and in vivo. In this chapter, we will describe methods utilized to purify SUMO conjugating enzymes and model substrates which can be used for analysis of SUMO conjugation in vitro. We will also describe methods to extract kinetic parameters during E3-dependent or E3-independent substrate conjugation. PMID:19107417

Incremental improvements to the trout S9 biotransformation assay

EPA Science Inventory

In vitro substrate depletion methods have been used in conjunction with computational models to predict biotransformation impacts on chemical accumulation by fish. There is a consistent trend, however, toward overestimation of measured chemical residues resulting from controlled...

Foot and mouth disease vaccine strain selection: Current approaches and future perspectives.

PubMed

Mahapatra, Mana; Parida, Satya

2018-06-27

Lack of cross protection between foot and mouth disease (FMD) virus (FMDV) serotypes as well as incomplete protection between some subtypes of FMDV affect the application of vaccine in the field. Further, the emergence of new variant FMD viruses periodically makes the existing vaccine inefficient. Consequently, periodical vaccine strain selection either by in vivo methods or in vitro methods become an essential requirement to enable utilisation of appropriate and efficient vaccines. Areas covered: Here we describe the cross reactivity of the existing vaccines with the global pool of circulating viruses and the putative selected vaccine strains for targeting protection against the two major circulating serotype O and A FMD viruses for East Africa, the Middle East, South Asia and South East Asia. Expert Commentary: Although in vivo cross protection studies are more appropriate methods for vaccine matching and selection than in vitro neutralisation test or ELISA, in the face of an outbreak both in vivo and in vitro methods of vaccine matching are not easy, and time consuming. The FMDV capsid contains all the immunogenic epitopes, and therefore vaccine strain prediction models using both capsid sequence and serology data will likely replace existing tools in the future.

Dry powder inhalers: An overview of the in vitro dissolution methodologies and their correlation with the biopharmaceutical aspects of the drug products.

PubMed

Velaga, Sitaram P; Djuris, Jelena; Cvijic, Sandra; Rozou, Stavroula; Russo, Paola; Colombo, Gaia; Rossi, Alessandra

2018-02-15

In vitro dissolution testing is routinely used in the development of pharmaceutical products. Whilst the dissolution testing methods are well established and standardized for oral dosage forms, i.e. tablets and capsules, there are no pharmacopoeia methods or regulatory requirements for testing the dissolution of orally inhaled powders. Despite this, a wide variety of dissolution testing methods for orally inhaled powders has been developed and their bio-relevance has been evaluated. This review provides an overview of the in vitro dissolution methodologies for dry inhalation products, with particular emphasis on dry powder inhalers, where the dissolution behavior of the respirable particles can have a role on duration and absorption of the drug. Dissolution mechanisms of respirable particles as well as kinetic models have been presented. A more recent biorelevant dissolution set-ups and media for studying inhalation biopharmaceutics were also reviewed. In addition, factors affecting interplay between dissolution and absorption of deposited particles in the context of biopharmaceutical considerations of inhalation products were examined. Copyright © 2017 Elsevier B.V. All rights reserved.

In vivo gene correction with targeted sequence substitution through microhomology-mediated end joining.

PubMed

Shin, Jeong Hong; Jung, Soobin; Ramakrishna, Suresh; Kim, Hyongbum Henry; Lee, Junwon

2018-07-07

Genome editing technology using programmable nucleases has rapidly evolved in recent years. The primary mechanism to achieve precise integration of a transgene is mainly based on homology-directed repair (HDR). However, an HDR-based genome-editing approach is less efficient than non-homologous end-joining (NHEJ). Recently, a microhomology-mediated end-joining (MMEJ)-based transgene integration approach was developed, showing feasibility both in vitro and in vivo. We expanded this method to achieve targeted sequence substitution (TSS) of mutated sequences with normal sequences using double-guide RNAs (gRNAs), and a donor template flanking the microhomologies and target sequence of the gRNAs in vitro and in vivo. Our method could realize more efficient sequence substitution than the HDR-based method in vitro using a reporter cell line, and led to the survival of a hereditary tyrosinemia mouse model in vivo. The proposed MMEJ-based TSS approach could provide a novel therapeutic strategy, in addition to HDR, to achieve gene correction from a mutated sequence to a normal sequence. Copyright © 2018 Elsevier Inc. All rights reserved.

Construction and histological analysis of a 3D human arterial wall model containing vasa vasorum using a layer-by-layer technique.

PubMed

Shima, Fumiaki; Narita, Hirokazu; Hiura, Ayami; Shimoda, Hiroshi; Akashi, Mitsuru

2017-03-01

There is considerable global demand for three-dimensional (3D) functional tissues which mimic our native organs and tissues for use as in vitro drug screening systems and in regenerative medicine. In particular, there has been an increasing number of patients who suffer from arterial diseases such as arteriosclerosis. As such, in vitro 3D arterial wall models that can evaluate the effects of novel medicines and a novel artificial graft for the treatment are required. In our previous study, we reported the rapid construction of 3D tissues by employing a layer-by-layer (LbL) technique and revealed their potential applications in the pharmaceutical fields and tissue engineering. In this study, we successfully constructed a 3D arterial wall model containing vasa vasorum by employing a LbL technique for the first time. The cells were coated with extracellular matrix nanofilms and seeded into a culture insert using a cell accumulation method. This model had a three-layered hierarchical structure: a fibroblast layer, a smooth muscle layer, and an endothelial layer, which resembled the native arterial wall. Our method could introduce vasa vasorum into a fibroblast layer in vitro and the 3D arterial wall model showed barrier function which was evaluated by immunostaining and transendothelial electrical resistance measurement. Furthermore, electron microscopy observations revealed that the vasa vasorum was composed of single-layered endothelial cells, and the endothelial tubes were surrounded by the basal lamina, which are known to promote maturation and stabilization in native blood capillaries. These models should be useful for tissue engineering, regenerative medicine, and pharmaceutical applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 814-823, 2017. © 2016 Wiley Periodicals, Inc.

An in vitro approach for lipolysis measurement using high-resolution mass spectrometry and partial least squares based analysis.

PubMed

Chang, Wen-Qi; Zhou, Jian-Liang; Li, Yi; Shi, Zi-Qi; Wang, Li; Yang, Jie; Li, Ping; Liu, Li-Fang; Xin, Gui-Zhong

2017-01-15

The elevation of free fatty acids (FFAs) has been regarded as a universal metabolic signature of excessive adipocyte lipolysis. Nowadays, in vitro lipolysis assay is generally essential for drug screening prior to the animal study. Here, we present a novel in vitro approach for lipolysis measurement combining UHPLC-Orbitrap and partial least squares (PLS) based analysis. Firstly, the calibration matrix was constructed by serial proportions of mixed samples (blended with control and model samples). Then, lipidome profiling was performed by UHPLC-Orbitrap, and 403 variables were extracted and aligned as dataset. Owing to the high resolution of Orbitrap analyzer and open source lipid identification software, 28 FFAs were further screened and identified. Based on the relative intensity of the screened FFAs, PLS regression model was constructed for lipolysis measurement. After leave-one-out cross-validation, ten principal components have been designated to build the final PLS model with excellent performances (RMSECV, 0.0268; RMSEC, 0.0173; R 2 , 0.9977). In addition, the high predictive accuracy (R 2  = 0.9907 and RMSEP = 0.0345) of the trained PLS model was also demonstrated using test samples. Finally, taking curcumin as a model compound, its antilipolytic effect on palmitic acid-induced lipolysis was successfully predicted as 31.78% by the proposed approach. Besides, supplementary evidences of curcumin induced modification in FFAs compositions as well as lipidome were given by PLS extended methods. Different from general biological assays, high resolution MS-based method provide more sophisticated information included in biological events. Thus, the novel biological evaluation model proposed here showed promising perspectives for drug evaluation or disease diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparative assessment of three in vitro exposure methods for combustion toxicity.

PubMed

Lestari, Fatma; Markovic, Boban; Green, Anthony R; Chattopadhyay, Gautam; Hayes, Amanda J

2006-01-01

A comparative assessment of three approaches for the use of human cells in vitro to investigate combustion toxicity was conducted. These included one indirect and two direct (passive and dynamic) exposure methods. The indirect method used an impinger system in which culture medium was used to trap the toxicants, whilst the direct exposure involved the use of a Horizontal Harvard Navicyte Chamber at the air/liquid interface. The cytotoxic effects of thermal decomposition products were assessed using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay (Promega) on a selection of human cells including: HepG2, A549 and skin fibroblasts. A small scale laboratory fire test using a vertical tube furnace was designed for the generation of combustion products. Polymethyl methacrylate (PMMA) was selected as a model polymer to study the cytotoxic effects of combustion products. NOAEC (no observable adverse effect concentration), IC10 (10% inhibitory concentration), IC50 (50% inhibitory concentration) and TLC (total lethal concentration) values were determined from dose response curves. Assessment using the NRU (neutral red uptake) and ATP (adenosine triphosphate) assays on human lung derived cells (A549) was also undertaken. Comparison between in vitro cytotoxicity results against published toxicity data for PMMA combustion and predicted LC50 (50% lethal concentration) values calculated from identified compounds using GCMS (gas chromatography mass spectrometry) was determined. The results suggested that the indirect exposure method did not appear to simulate closely exposure via inhalation, whilst exposure at the air/liquid interface by using the dynamic method proved to be a more representative method of human inhalation. This exposure method may be a potential system for in vitro cytotoxicity testing in combustion toxicity. Copyright 2005 John Wiley & Sons, Ltd.

An in vitro method to test the safety and efficacy of low-level laser therapy (LLLT) in the healing of a canine skin model.

PubMed

Gagnon, Dominique; Gibson, Thomas W G; Singh, Ameet; zur Linden, Alex R; Kazienko, Jaimie E; LaMarre, Jonathan

2016-04-08

Low-level laser therapy (LLLT) has been used clinically as a treatment modality for a variety of medical conditions including wound-healing processes. It is an attractive and emerging method to enhance wound healing and improve clinical outcomes both in human and veterinary medicine. Despite the fact that the use of LLLT continues to gain in popularity, there is no universally accepted theory that defends all its cellular effects and beneficial biological processes in tissue repair. The present study was designed to evaluate the effect of LLLT on cellular migration and proliferation of cultured canine epidermal keratinocytes (CPEK) in an in vitro wound healing model. Keratinocyte migration and proliferation were assessed using a scratch migration assay and a proliferation assay, respectively. Fifteen independent replicates were performed for each assay. Canine epidermal keratinocyte cells exposed to LLLT with 0.1, 0.2, and 1.2 J/cm(2) migrated significantly more rapidly (p < 0.03) and showed significantly higher rates of proliferation (p < 0.0001) compared to non-irradiated cells cultured in the same medium and cells exposed to the higher energy dose of 10 J/cm(2). Irradiation with 10 J/cm(2) was characterized by decreased cellular migration and proliferation. These results revealed that LLLT has a measurable, dose-dependent effect on two different aspects of keratinocyte biology in vitro. In this in vitro wound-healing model, LLLT increased cellular migration and proliferation at doses of 0.1, 0.2, and 1.2 J/cm(2) while exposure to 10 J/cm(2) decreased cellular migration and proliferation. These data suggest that the beneficial effects of LLLT in vivo may be due, in part, to effects on keratinocyte behavior.

Reconstituted human gingival epithelium: nonsubmerged in vitro model.

PubMed

Delcourt-Huard, A; Corlu, A; Joffre, A; Magloire, H; Bonnaure-Mallet, M

1997-01-01

Many studies have shown that human gingival keratinocytes grown in submerged culture fail to attain optimal differentiation. This study reports an in vitro culture system for oral gingival epithelial cells, in which they are grown at the air-liquid interface, on polycarbonate inserts, in the presence of an NIH-3T3 feeder layer. This model was compared with two submerged culture methods for gingival keratinocytes, on type 1 collagen gel and on an NIH-3T3 feeder layer. Transmission electron microscopy showed an advanced level of stratification (over six layers of cells) for cultures grown at the air-liquid interface. Immunofluorescence and electrophoretic patterns showed the presence of cytokeratins 10 and 11 in cytoskeletal protein extracts of these cultured keratinocytes. In this air-liquid interface culture model, in the presence of NIH-3T3 feeder cells, keratinocytes can achieve an advanced level of stratification and differentiation and a resemblance to in vivo gingiva. The obtention of a highly differentiated epithelium will permit in vitro pharmacological studies and studies on the biocompatability of certain alloys with the superficial periodontium; it will also provide grafts for patients undergoing periodontal surgery.

Accelerated in-vitro release testing methods for extended-release parenteral dosage forms.

PubMed

Shen, Jie; Burgess, Diane J

2012-07-01

This review highlights current methods and strategies for accelerated in-vitro drug release testing of extended-release parenteral dosage forms such as polymeric microparticulate systems, lipid microparticulate systems, in-situ depot-forming systems and implants. Extended-release parenteral dosage forms are typically designed to maintain the effective drug concentration over periods of weeks, months or even years. Consequently, 'real-time' in-vitro release tests for these dosage forms are often run over a long time period. Accelerated in-vitro release methods can provide rapid evaluation and therefore are desirable for quality control purposes. To this end, different accelerated in-vitro release methods using United States Pharmacopeia (USP) apparatus have been developed. Different mechanisms of accelerating drug release from extended-release parenteral dosage forms, along with the accelerated in-vitro release testing methods currently employed are discussed. Accelerated in-vitro release testing methods with good discriminatory ability are critical for quality control of extended-release parenteral products. Methods that can be used in the development of in-vitro-in-vivo correlation (IVIVC) are desirable; however, for complex parenteral products this may not always be achievable. © 2012 The Authors. JPP © 2012 Royal Pharmaceutical Society.

Accelerated in vitro release testing methods for extended release parenteral dosage forms

PubMed Central

Shen, Jie; Burgess, Diane J.

2012-01-01

Objectives This review highlights current methods and strategies for accelerated in vitro drug release testing of extended release parenteral dosage forms such as polymeric microparticulate systems, lipid microparticulate systems, in situ depot-forming systems, and implants. Key findings Extended release parenteral dosage forms are typically designed to maintain the effective drug concentration over periods of weeks, months or even years. Consequently, “real-time” in vitro release tests for these dosage forms are often run over a long time period. Accelerated in vitro release methods can provide rapid evaluation and therefore are desirable for quality control purposes. To this end, different accelerated in vitro release methods using United States Pharmacopoeia (USP) apparatus have been developed. Different mechanisms of accelerating drug release from extended release parenteral dosage forms, along with the accelerated in vitro release testing methods currently employed are discussed. Conclusions Accelerated in vitro release testing methods with good discriminatory ability are critical for quality control of extended release parenteral products. Methods that can be used in the development of in vitro-in vivo correlation (IVIVC) are desirable, however for complex parenteral products this may not always be achievable. PMID:22686344

Effect of a triclosan/PVM/MA copolymer/fluoride dentifrice on volatile sulfur compounds in vitro.

PubMed

Pilch, S; Williams, M I; Cummins, D

2005-01-01

The objective of the investigation was to document the in vitro efficacy of a triclosan/PVM/MA copolymer/fluoride (TCF) dentifrice against the formation of volatile sulfur compounds (VSC) as well as the growth of H2S-producing bacteria. Clinical studies using organoleptic judges, gas chromatography, or a portable sulfide monitor have generally been employed in the assessment of treatments for the control of oral malodor. However, these studies are not appropriate for screening purposes because of the expense and time required. An in vitro method was developed for the purpose of screening new compounds, agents or formulations for their ability to control VSC formation and for determining bio-equivalence of efficacy when implementing changes in existing formulations. The method combines basic microbiological methods, dynamic flow cell techniques and head space analysis. The in vitro VSC method was validated by comparing the efficacy of two dentifrices containing TCF with a control fluoride dentifrice as the TCF products have been clinically proven to control oral malodor. In the validation studies, the TCF-containing dentifrices were significantly better (P < 0.05) than the control dentifrice in inhibiting VSC formation and reducing H(2)S-producing bacteria. For example, when compared with baseline, the TCF dentifrices reduced VSC formation between 42 and 49% compared with the control dentifrice which reduced VSC formation 3%. There was no significant difference (P > 0.05) between the two TCF dentifrice formulations. Using an in vitro breath VSC model, it has been demonstrated that two variants of a dentifrice containing triclosan, PVM/MA copolymer and fluoride have efficacy that is significantly better than a control fluoridated dentifrice and that there is no significant difference between the triclosan/PVM/MA copolymer/fluoride dentifrice variants.

Comparative evaluation of the efficacy of a herbal mouthwash and chlorhexidine mouthwash on select periodontal pathogens: An in vitro and ex vivo study

PubMed Central

Pathan, Multazim Muradkhan; Bhat, Kishore Gajanan; Joshi, Vinayak Mahableshwar

2017-01-01

Background: Several herbal mouthwash and herbal extracts have been tested in vitro and in vivo in search of a suitable adjunct to mechanical therapy for long-term use. In this study, we aimed to look at the antimicrobial effect of the herbal mouthwash and chlorhexidine (CHX) mouthwash on select organisms in in vitro test and an ex vivo model. Materials and Methods: The antimicrobial effects were determined against standard strains of bacteria that are involved in different stages of periodontal diseases. The in vitro tests included determination of minimum inhibitory concentration (MIC) using broth dilution and agar diffusion. In the ex vivo part of the study supragingival dental plaque were obtained from 20 periodontally healthy adult volunteers. Descriptive analysis was done for the entire quantitative and qualitative variable recorded. Results: The MIC by broth dilution method found no statistically significant difference between the mouthwashes. The agar dilution method showed CHX was more effective as compared to the herbal mouthwash against standard strains of Streptococcus mutans, Streptococcus sanguinis, and Aggregatibacter actinomycetemcomitans. However, no difference was observed between the mouthwashes for Porphyromonas, Pseudomonas aeruginosa, and Fusobacterium nucleatum. The ex vivo results conclude that none of the selected mouthwashes were statistically significantly different from each other. Conclusion: In the present study, CHX showed higher levels of antimicrobial action than the herbal mouthwash against bacterial species. The results reinforce the earlier findings that the in vitro testing is sensitive to methods and due diligence is needed when extrapolating the data for further use. However, long-term use and in vivo effectiveness against the periopathogens need to be tested in well-planned clinical trials. PMID:29456300

In vitro and in vivo comparisons of constant resistance AC iontophoresis and DC iontophoresis.

PubMed

Li, S Kevin; Higuchi, William I; Zhu, Honggang; Kern, Steven E; Miller, David J; Hastings, Matthew S

2003-09-04

A previous in vitro constant electrical resistance alternating current (AC) iontophoresis study with human epidermal membrane (HEM) and a model neutral permeant has shown less inter- and intra-sample variability in iontophoretic transport relative to conventional constant direct current (DC) iontophoresis. The objectives of the present study were to address the following questions. (1) Can the skin electrical resistance be maintained at a constant level by AC in humans in vivo? (2) Are the in vitro data with HEM representative of those in vivo? (3) Does constant skin resistance AC iontophoresis have less inter- and intra-sample variability than conventional constant current DC iontophoresis in vivo? (4) What are the electrical and the barrier properties of skin during iontophoresis in vivo? In the present study, in vitro HEM experiments were carried out with the constant resistance AC and the conventional constant current DC methods using mannitol and glucose as the neutral model permeants. In vivo human experiments were performed using glucose as the permeant with a constant skin resistance AC only protocol and two conventional constant current DC methods (continuous constant current DC and constant current DC with its polarity alternated every 10 min with a 3:7 on:off duty cycle). Constant current DC iontophoresis was conducted with commercial constant current DC devices, and constant resistance AC iontophoresis was carried out by reducing and maintaining the skin resistance at a constant target value with AC supplied from a function generator. This study shows that (1) skin electrical resistance can be maintained at a constant level during AC iontophoresis in vivo; (2) HEM in vitro and human skin in vivo demonstrate similar electrical and barrier properties, and these properties are consistent with our previous findings; (3) there is general qualitative and semi-quantitative agreement between the HEM data in vitro and human skin data in vivo; and (4) constant skin resistance AC iontophoresis generally provides less inter- and intra-subject variability than conventional constant current DC.

Alternative Test Methods for Developmental Neurotoxicity: A ...

EPA Pesticide Factsheets

Exposure to environmental contaminants is well documented to adversely impact the development of the nervous system. However, the time, animal and resource intensive EPA and OECD testing guideline methods for developmental neurotoxicity (DNT) are not a viable solution to characterizing potential chemical hazards for the thousands of untested chemicals currently in commerce. Thus, research efforts over the past decade have endeavored to develop cost-effective alternative DNT testing methods. These efforts have begun to generate data that can inform regulatory decisions. Yet there are major challenges to both the acceptance and use of this data. Major scientific challenges for DNT include development of new methods and models that are “fit for purpose”, development of a decision-use framework, and regulatory acceptance of the methods. It is critical to understand that use of data from these methods will be driven mainly by the regulatory problems being addressed. Some problems may be addressed with limited datasets, while others may require data for large numbers of chemicals, or require the development and use of new biological and computational models. For example mechanistic information derived from in vitro DNT assays can be used to inform weight of evidence (WoE) or integrated approaches to testing and assessment (IATA) approaches for chemical-specific assessments. Alternatively, in vitro data can be used to prioritize (for further testing) the thousands

SU-F-T-105: The Predicted Radiation Response of Non-Small Cell Lung Cancer for SRS, SBRT and HDR Brachytherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Z; Feng, Y; Lo, S

2016-06-15

Purpose: The data on the α/β ratio of non-small cell lung cancer (NSCLC) is scarce in the literature. We have previously proposed a generalized LQ (gLQ) model to address the high dose dilemma of the LQ model. In this study, we applied the gLQ model to both the patients and in vitro cell irradiation data treated with a large range of doses, and investigated the α/β ratio in NSCLC. Methods: 150 patients with T1T2 and non-T1T2 stages were treated with stereotactic body radiotherapy (SBRT). In vitro datasets of 14 NSCLC cell lines from the National Cancer Institute published in Eurmore » J Cancer Clin Oncol. 25(3):527–534 (1989) and 7 NSCLC cell lines published in Cancer Res 57:4285–300 (1997) were included. The gLQ model was used to fit datasets. The least χ2 method was adopted to determine the goodness of fit. Errors of the model parameters were determined by propagating minimal χ2. The α/β ratios from both the patients and these in vitro NSCLC cell lines were obtained. Results: The average of α/β ratios for T1T2 and non-T1T2 NSCLC was 1.45 Gy. The same type of cell lines irradiated with different modalities but almost the same dose rate yielded approximately the same α/β ratio. The average of α/β ratios for NSCLC cell lines in this study was 5.45 Gy. Conclusion: The difference in the α/β ratios between the patients and in vitro cell data is expected and the lower α/β ratio for patients suggests the higher radiosensitivity, which could be associated with higher tumor perfusion or other tumor microenvironmental effects. The α/β ratios derived from the gLQ model can be used in high dose regions or high fraction sizes and are useful to extend our clinical experience accumulated from conversional low-dose fractionation to high dose irradiation schedules.« less

In vitro and in vivo evaluation of the bioactivity of hydroxyapatite-coated polyetheretherketone biocomposites created by cold spray technology.

PubMed

Lee, Jae Hyup; Jang, Hae Lin; Lee, Kyung Mee; Baek, Hae-Ri; Jin, Kyoungsuk; Hong, Kug Sun; Noh, Jun Hong; Lee, Hyun-Kyung

2013-04-01

Polyetheretherketone (PEEK) is a material that is widely used in medicine because its mechanical properties show excellent similarity to those of human bone. However, because it is bioinert, PEEK shows limited ability to bind to natural bone tissue. Here, we applied a cold spray method to make a hydroxyapatite (HA)-coated PEEK hybrid material and evaluated its osteointegration in vitro and in vivo. With the cold spray method, the HA coating formed a homogeneous layer and adhered strongly to the PEEK disk implant. When the material was tested in vitro, early cell adhesion and viability improved. Alkaline phosphatase (ALP) activity and calcium concentration were also higher in cells cultured on HA-coated PEEK disks. In addition, the expression of osteoblast differentiation markers, such as ALP, bone sialoprotein and runt-related transcription factor 2, increased in these cells. For the in vivo test, we designed and implanted HA-coated PEEK cylinders into a rabbit ilium model by the press-fit method. The bone-implant contact ratio, trabecular number and trabecular thickness were determined using either three-dimensional microcomputed tomography or general two-dimensional histomorphometric analysis. This report demonstrates that the HA coating on the PEEK implant added with the cold spray method increased biocompatibility in vitro and promoted osteointegration in vivo, which suggests that the HA coating may improve the biofunctionality of various medical devices used in clinical applications. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Development of a reservoir type prolonged release system with felodipine via simplex methodology

PubMed Central

IOVANOV, RAREŞ IULIU; TOMUŢĂ, IOAN; LEUCUŢA, SORIN EMILIAN

2016-01-01

Background and aims Felodipine is a dihydropyridine calcium antagonist that presents good characteristics to be formulated as prolonged release preparations. The aim of the study was the formulation and in vitro characterization of a reservoir type prolonged release system with felodipine, over a 12 hours period using the Simplex method. Methods The first step of the Simplex method was to study the influence of the granules coating method on the felodipine release. Furthermore the influence of the coating polymer type, the percent of the coating polymer and the percent of pore forming agent in the coating on the felodipine release were studied. Afterwards these two steps of the experimental design the percent of Surelease applied on the felodipine loaded granules and the percent of pore former in the polymeric coating formulation variables were studied. The in vitro dissolution of model drug was performed in phosphate buffer solution (pH 6.5) with 1% sodium lauryl sulfate. The released drug quantification was done using an HPLC method. The release kinetics of felodipine from the final granules was assessed using different mathematical models. Results A 12 hours release was achieved using granules with the size between 315–500 μm coated with 45% Surelease with different pore former ratios in the coating via the top-spray method. Conclusion We have prepared prolonged release coated granules with felodipine using a fluid bed system based on the Simplex method. The API from the studied final formulations was released over a 12 hours period and the release kinetics of the model drug substance from the optimized preparations fitted best the Higuchi and Peppas kinetic models. PMID:27004036

A numerical model to reproduce squeaking of ceramic-on-ceramic total hip arthroplasty. Influence of design and material.

PubMed

Piriou, P; Ouenzerfi, G; Migaud, H; Renault, E; Massi, F; Serrault, M

2016-06-01

Modern ceramic (CoC) bearings for hip arthroplasty (THA) have been used in younger patients who expect improved survivorship. However, audible squeaking produced by the implant is an annoying complication. Previous numerical simulations were not able to accurately reproduce in vitro and in vivo observations. Therefore, we developed a finite element model to: (1) reproduce in vitro squeaking and validate the model by comparing it with in vivo recordings, (2) determine why there are differences between in vivo and in vitro squeaking frequencies, (3) identify the stem's role in this squeaking, (4) predict which designs and materials are more likely to produce squeaking. A CoC THA numerical model can be developed that reproduces the squeaking frequencies observed in vivo. Numerical methods (finite element analysis [ANSYS]) and experimental methods (using a non-lubricated simulated hip with a cementless 32mm CoC THA) were developed to reproduce squeaking. Numerical analysis was performed to identify the frequencies that cause vibrations perceived as an acoustic emission. The finite element analysis (FEA) model was enhanced by adjusting periprosthetic bone and soft tissue elements in order to reproduce the squeaking frequencies recorded in vivo. A numerical method (complex eigenvalue analysis) was used to find the acoustic frequencies of the squeaking noise. The frequencies obtained from the model and the hip simulator were compared to those recorded in vivo. The numerical results were validated by experiments with the laboratory hip simulator. The frequencies obtained (mean 2790Hz with FEA, 2755Hz with simulator, decreasing to 1759Hz when bone and soft tissue were included in the FEA) were consistent with those of squeaking hips recorded in vivo (1521Hz). The cup and ceramic insert were the source of the vibration, but had little influence on the diffusion of the noise required to make the squeaking audible to the human ear. The FEA showed that diffusion of squeaking was due to an unstable vibration of the stem during frictional contact. The FEA predicted a higher rate of squeaking (at a lower coefficient of friction) when TZMF™ alloy is used instead of Ti6Al4V and when an anatomic press-fit stem is used instead of straight self-locking designs. The current FEA model is reliable; it can be used to assess various stem designs and alloys to predict the different rates of squeaking that certain stems will likely produce. Level IV in vitro study. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

The beneficial effects of cumulus cells and oocyte-cumulus cell gap junctions depends on oocyte maturation and fertilization methods in mice.

PubMed

Zhou, Cheng-Jie; Wu, Sha-Na; Shen, Jiang-Peng; Wang, Dong-Hui; Kong, Xiang-Wei; Lu, Angeleem; Li, Yan-Jiao; Zhou, Hong-Xia; Zhao, Yue-Fang; Liang, Cheng-Guang

2016-01-01

Cumulus cells are a group of closely associated granulosa cells that surround and nourish oocytes. Previous studies have shown that cumulus cells contribute to oocyte maturation and fertilization through gap junction communication. However, it is not known how this gap junction signaling affects in vivo versus in vitro maturation of oocytes, and their subsequent fertilization and embryonic development following insemination. Therefore, in our study, we performed mouse oocyte maturation and insemination using in vivo- or in vitro-matured oocyte-cumulus complexes (OCCs, which retain gap junctions between the cumulus cells and the oocytes), in vitro-matured, denuded oocytes co-cultured with cumulus cells (DCs, which lack gap junctions between the cumulus cells and the oocytes), and in vitro-matured, denuded oocytes without cumulus cells (DOs). Using these models, we were able to analyze the effects of gap junction signaling on oocyte maturation, fertilization, and early embryo development. We found that gap junctions were necessary for both in vivo and in vitro oocyte maturation. In addition, for oocytes matured in vivo, the presence of cumulus cells during insemination improved fertilization and blastocyst formation, and this improvement was strengthened by gap junctions. Moreover, for oocytes matured in vitro, the presence of cumulus cells during insemination improved fertilization, but not blastocyst formation, and this improvement was independent of gap junctions. Our results demonstrate, for the first time, that the beneficial effect of gap junction signaling from cumulus cells depends on oocyte maturation and fertilization methods.

Advances and perspectives in in vitro human gut fermentation modeling.

PubMed

Payne, Amanda N; Zihler, Annina; Chassard, Christophe; Lacroix, Christophe

2012-01-01

The gut microbiota is a highly specialized organ containing host-specific assemblages of microbes whereby metabolic activity directly impacts human health and disease. In vitro gut fermentation models present an unmatched opportunity of performing studies frequently challenged in humans and animals owing to ethical concerns. Multidisciplinary systems biology analyses supported by '-omics' platforms remain widely neglected in the field of in vitro gut fermentation modeling but are key to advancing the significance of these models. Model-driven experimentation using a combination of in vitro gut fermentation and in vitro human cell models represent an advanced approach in identifying complex host-microbe interactions and niches central to gut fermentation processes. The aim of this review is to highlight the advances and challenges exhibited by in vitro human gut fermentation modeling. Copyright © 2011 Elsevier Ltd. All rights reserved.

Verification of finite element analysis of fixed partial denture with in vitro electronic strain measurement.

PubMed

Wang, Gaoqi; Zhang, Song; Bian, Cuirong; Kong, Hui

2016-01-01

The purpose of the study was to verify the finite element analysis model of three-unite fixed partial denture with in vitro electronic strain analysis and analyze clinical situation with the verified model. First, strain gauges were attached to the critical areas of a three-unit fixed partial denture. Strain values were measured under 300 N load perpendicular to the occlusal plane. Secondly, a three-dimensional finite element model in accordance with the electronic strain analysis experiment was constructed from the scanning data. And the strain values obtained by finite element analysis and in vitro measurements were compared. Finally, the clinical destruction of the fixed partial denture was evaluated with the verified finite element analysis model. There was a mutual agreement and consistency between the finite element analysis results and experimental data. The finite element analysis revealed that failure will occur in the veneer layer on buccal surface of the connector under occlusal force of 570 N. The results indicate that the electronic strain analysis is an appropriate and cost saving method to verify the finite element model. The veneer layer on buccal surface of the connector is the weakest area in the fixed partial denture. Copyright © 2015 Japan Prosthodontic Society. Published by Elsevier Ltd. All rights reserved.

In vitro validation of a novel mechanical model for testing the anchorage capacity of pedicle screws using physiological load application.

PubMed

Liebsch, Christian; Zimmermann, Julia; Graf, Nicolas; Schilling, Christoph; Wilke, Hans-Joachim; Kienle, Annette

2018-01-01

Biomechanical in vitro tests analysing screw loosening often include high standard deviations caused by high variabilities in bone mineral density and pedicle geometry, whereas standardized mechanical models made of PU foam often do not integrate anatomical or physiological boundary conditions. The purpose of this study was to develop a most realistic mechanical model for the standardized and reproducible testing of pedicle screws regarding the resistance against screw loosening and the holding force as well as to validate this model by in vitro experiments. The novel mechanical testing model represents all anatomical structures of a human vertebra and is consisting of PU foam to simulate cancellous bone, as well as a novel pedicle model made of short carbon fibre filled epoxy. Six monoaxial cannulated pedicle screws (Ø6.5 × 45mm) were tested using the mechanical testing model as well as human vertebra specimens by applying complex physiological cyclic loading (shear, tension, and bending; 5Hz testing frequency; sinusoidal pulsating forces) in a dynamic materials testing machine with stepwise increasing load after each 50.000 cycles (100.0N shear force + 20.0N per step, 51.0N tension force + 10.2N per step, 4.2Nm bending moment + 0.8Nm per step) until screw loosening was detected. The pedicle screw head was fixed on a firmly clamped rod while the load was applied in the vertebral body. For the in vitro experiments, six human lumbar vertebrae (L1-3, BMD 75.4 ± 4.0mg/cc HA, pedicle width 9.8 ± 0.6mm) were tested after implanting pedicle screws under X-ray control. Relative motions of pedicle screw, specimen fixture, and rod fixture were detected using an optical motion tracking system. Translational motions of the mechanical testing model experiments in the point of load introduction (0.9-2.2mm at 240N shear force) were reproducible within the variation range of the in vitro experiments (0.6-3.5mm at 240N shear force). Screw loosening occurred continuously in each case between 140N and 280N, while abrupt failures of the specimen were observed only in vitro. In the mechanical testing model, no translational motion was detected in the screw entry point, while in vitro, translational motions of up to 2.5mm in inferior direction were found, leading to a slight shift of the centre of rotation towards the screw tip. Translational motions of the screw tip of about 5mm in superior direction were observed both in vitro and in the mechanical testing model, while they were continuous in the mechanical testing model and rapidly increasing after screw loosening initiation in vitro. The overall pedicle screw loosening characteristics were qualitatively and quantitatively similar between the mechanical testing model and the human vertebral specimens as long as there was no translation of the screw at the screw entrance point. Therefore, the novel mechanical testing model represents a promising method for the standardized testing of pedicle screws regarding screw loosening for cases where the screw rotates around a point close to the screw entry point. Copyright © 2017 Elsevier Ltd. All rights reserved.

Reduced injection pressures using a compressed air injection technique (CAIT): an in vitro study.

PubMed

Tsui, Ban C H; Knezevich, Mark P; Pillay, Jennifer J

2008-01-01

High injection pressures have been associated with intraneural injection and persistent neurological injury in animals. Our objective was to test whether a reported simple compressed air injection technique (CAIT) would limit the generation of injection pressures to below a suggested 1,034 mm Hg limit in an in vitro model. After ethics board approval, 30 consenting anesthesiologists injected saline into a semiclosed system. Injection pressures using 30 mL syringes connected to a 22 gauge needle and containing 20 mL of saline were measured for 60 seconds using: (1) a typical "syringe feel" method, and (2) CAIT, thereby drawing 10 mL of air above the saline and compressing this to 5 mL prior to and during injections. All anesthesiologists performed the syringe feel method before introduction and demonstration of CAIT. Using CAIT, no anesthesiologist generated pressures above 1,034 mm Hg, while 29 of 30 produced pressures above this limit at some time using the syringe feel method. The mean pressure using CAIT was lower (636 +/- 71 vs. 1378 +/- 194 mm Hg, P = .025), and the syringe feel method resulted in higher peak pressures (1,875 +/- 206 vs. 715 +/- 104 mm Hg, P = .000). This study demonstrated that CAIT can effectively keep injection pressures under 1,034 mm Hg in this in vitro model. Animal and clinical studies will be needed to determine whether CAIT will allow objective, real-time pressure monitoring. If high pressure injections are proven to contribute to nerve injury in humans, this technique may have the potential to improve the safety of peripheral nerve blocks.

Particokinetics: computational analysis of the superparamagnetic iron oxide nanoparticles deposition process

PubMed Central

Cárdenas, Walter HZ; Mamani, Javier B; Sibov, Tatiana T; Caous, Cristofer A; Amaro, Edson; Gamarra, Lionel F

2012-01-01

Background Nanoparticles in suspension are often utilized for intracellular labeling and evaluation of toxicity in experiments conducted in vitro. The purpose of this study was to undertake a computational modeling analysis of the deposition kinetics of a magnetite nanoparticle agglomerate in cell culture medium. Methods Finite difference methods and the Crank–Nicolson algorithm were used to solve the equation of mass transport in order to analyze concentration profiles and dose deposition. Theoretical data were confirmed by experimental magnetic resonance imaging. Results Different behavior in the dose fraction deposited was found for magnetic nanoparticles up to 50 nm in diameter when compared with magnetic nanoparticles of a larger diameter. Small changes in the dispersion factor cause variations of up to 22% in the dose deposited. The experimental data confirmed the theoretical results. Conclusion These findings are important in planning for nanomaterial absorption, because they provide valuable information for efficient intracellular labeling and control toxicity. This model enables determination of the in vitro transport behavior of specific magnetic nanoparticles, which is also relevant to other models that use cellular components and particle absorption processes. PMID:22745539

Toward a CRISPR Picture: Use of CRISPR/Cas9 to Model Diseases in Human Stem Cells In Vitro.

PubMed

Freiermuth, Jamie L; Powell-Castilla, Ian J; Gallicano, G Ian

2018-01-01

Human induced pluripotent stem cells (iPSCs) can be differentiated into any cell in the body unlocking enormous research potential. Combined with the recent discovery of CRISPR/Cas9 endonucleases in bacteria and their modification for use in biomedical research, these methods have the potential to revolutionize the field of genetic engineering and open the door to generating in vitro models that more closely resemble the in vivo system than ever before. Use of CRISPR/Cas9 has created a whirlwind within the scientific community in the last few years, as the race to move beyond just disease analysis and toward the goal of gene and cell therapy moves further. This review will detail the CRISPR/Cas9 method and its use in stem cells as well as highlight recent studies that demonstrate its use in creating robust disease models. Finally, recent results and current controversies in the field are reviewed and lingering challenges to further development are explored. J. Cell. Biochem. 119: 62-68, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Phototoxicity to sulphonamide-derived oral antidiabetics and diuretics: comparative in-vitro and in-vivo investigations

NASA Astrophysics Data System (ADS)

Selvaag, Edgar; Anholt, Helle; Moan, Johan; Thune, Per

1997-12-01

Seven oral antidiabetics (chlorpropamide, glibenclamide, glipizide, gliquidone, glymidine, tolazamide, and tolbutamide), and 14 diuretics (bemetizide, bendroflumethiazide, benzylhydrochlorothiazide, bumetanide, butizide, chlortalidone, furosemide, hydrochlorothiazide, hydroflumethiazide, indapamide, piretanide, polythiazide, trichlormethiazide, and xipamide) were investigated for potential phototoxicity in vitro using a cell culture model and in vivo in hairless mice. After exposure to broad band UVA, the majority of the substances tested in vitro yielded phototoxic action leading to loss of culture forming ability. In vivo, all tested substances induced edema or ulceration, and lead to a significant increase in skin fold thickness of the mouse skin. In all a number of substances not described to induce clinical photosensitivity nor phototoxicity in vitro or in vivo were detected in our testing. In determining potential photosensitizers, it seems important to utilize different test methods, as not all substances will exhibit action in a given assay.

In Vitro Engineering of Vascularized Tissue Surrogates

PubMed Central

Sakaguchi, Katsuhisa; Shimizu, Tatsuya; Horaguchi, Shigeto; Sekine, Hidekazu; Yamato, Masayuki; Umezu, Mitsuo; Okano, Teruo

2013-01-01

In vitro scaling up of bioengineered tissues is known to be limited by diffusion issues, specifically a lack of vasculature. Here, we report a new strategy for preserving cell viability in three-dimensional tissues using cell sheet technology and a perfusion bioreactor having collagen-based microchannels. When triple-layer cardiac cell sheets are incubated within this bioreactor, endothelial cells in the cell sheets migrate to vascularize in the collagen gel, and finally connect with the microchannels. Medium readily flows into the cell sheets through the microchannels and the newly developed capillaries, while the cardiac construct shows simultaneous beating. When additional triple-layer cell sheets are repeatedly layered, new multi-layer construct spontaneously integrates and the resulting construct becomes a vascularized thick tissue. These results confirmed our method to fabricate in vitro vascularized tissue surrogates that overcomes engineered-tissue thickness limitations. The surrogates promise new therapies for damaged organs as well as new in vitro tissue models. PMID:23419835

In vitro simulator with numerical stress analysis for evaluation of stent-assisted coiling embolization in cerebral aneurysm treatments.

PubMed

Shi, Chaoyang; Kojima, Masahiro; Tercero, Carlos; Najdovski, Zoran; Ikeda, Seiichi; Fukuda, Toshio; Arai, Fumihito; Negoro, Makoto

2014-12-01

There are several complications associated with Stent-assisted Coil Embolization (SACE) in cerebral aneurysm treatments, due to damaging operations by surgeons and undesirable mechanical properties of stents. Therefore, it is necessary to develop an in vitro simulator that provides both training and research for evaluating the mechanical properties of stents. A new in vitro simulator for three-dimensional digital subtraction angiography was constructed, followed by aneurysm models fabricated with new materials. Next, this platform was used to provide training and to conduct photoelastic stress analysis to evaluate the SACE technique. The average interaction stress increasingly varied for the two different stents. Improvements for the Maximum-Likelihood Expectation-Maximization method were developed to reconstruct cross-sections with both thickness and stress information. The technique presented can improve a surgeon's skills and quantify the performance of stents to improve mechanical design and classification. This method can contribute to three-dimensional stress and volume variation evaluation and assess a surgeon's skills. Copyright © 2013 John Wiley & Sons, Ltd.

Modeling Hematopoiesis and Responses to Radiation Countermeasures in a Bone Marrow-on-a-Chip.

PubMed

Torisawa, Yu-Suke; Mammoto, Tadanori; Jiang, Elisabeth; Jiang, Amanda; Mammoto, Akiko; Watters, Alexander L; Bahinski, Anthony; Ingber, Donald E

2016-05-01

Studies on hematopoiesis currently rely on animal models because in vitro culture methods do not accurately recapitulate complex bone marrow physiology. We recently described a bone marrow-on-a-chip microfluidic device that enables the culture of living hematopoietic bone marrow and mimics radiation toxicity in vitro. In the present study, we used this microdevice to demonstrate continuous blood cell production in vitro and model bone marrow responses to potential radiation countermeasure drugs. The device maintained mouse hematopoietic stem and progenitor cells in normal proportions for at least 2 weeks in culture. Increases in the number of leukocytes and red blood cells into the microfluidic circulation also could be detected over time, and addition of erythropoietin induced a significant increase in erythrocyte production. Exposure of the bone marrow chip to gamma radiation resulted in reduction of leukocyte production, and treatment of the chips with two potential therapeutics, granulocyte-colony stimulating factor or bactericidal/permeability-increasing protein (BPI), induced significant increases in the number of hematopoietic stem cells and myeloid cells in the fluidic outflow. In contrast, BPI was not found to have any effect when analyzed using static marrow cultures, even though it has been previously shown to accelerate recovery from radiation-induced toxicity in vivo. These findings demonstrate the potential value of the bone marrow-on-a-chip for modeling blood cell production, monitoring responses to hematopoiesis-modulating drugs, and testing radiation countermeasures in vitro.

Implant Impression Techniques for the Edentulous Jaw: A Summary of Three Studies.

PubMed

Stimmelmayr, Michael; Beuer, Florian; Edelhoff, Daniel; Güth, Jan-Frederik

2016-02-01

Precise implant-supported restorations require accurate impressions. Transfer, pick-up, and splinted pick-up are commonly used techniques. Several in vitro studies have compared these impression techniques; however, all studies used mechanical evaluation methods. The purpose of this study was to compare the discrepancies of these impression techniques digitally in vitro and in vivo. Four dental implants were inserted in ten polymer mandibular models bilaterally in the regions of the first molars and canines. Three different impressions were made of each model and the models (original and stone casts) were scanned and digitized. Clinically, four implants were inserted in ten edentulous jaws; transfer and splinted pick-up impressions were made. With inspection software, discrepancies between the different impressions were calculated. The mean discrepancies in the in vitro study of the original polymer model to stone casts were 124 ± 34 μm for the transfer type, 116 ± 46 μm for the pick-up type, and 80 ± 25 μm for the splinted pick-up type, resulting in a mean discrepancy between the transfer and splinted pick-up type of 44 μm (124 - 80 μm). Clinically, the mean discrepancy between these two impression techniques was 280 μm. The differing results between the transfer and splinted pick-up techniques of in vitro and in vivo data showed the need for clinical data; however, splinted pick-up impressions seemed to produce the most precise results. © 2015 by the American College of Prosthodontists.

Development of achiral and chiral 2D HPLC methods for analysis of albendazole metabolites in microsomal fractions using multivariate analysis for the in vitro metabolism.

PubMed

Belaz, Kátia Roberta A; Pereira-Filho, Edenir Rodrigues; Oliveira, Regina V

2013-08-01

In this work, the development of two multidimensional liquid chromatography methods coupled to a fluorescence detector is described for direct analysis of microsomal fractions obtained from rat livers. The chiral multidimensional method was then applied for the optimization of the in vitro metabolism of albendazole by experimental design. Albendazole was selected as a model drug because of its anthelmintics properties and recent potential for cancer treatment. The development of two fully automated achiral-chiral and chiral-chiral high performance liquid chromatography (HPLC) methods for the determination of albendazole (ABZ) and its metabolites albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO2) and albendazole 2-aminosulphone (ABZ-SO2NH2) in microsomal fractions are described. These methods involve the use of a phenyl (RAM-phenyl-BSA) or octyl (RAM-C8-BSA) restricted access media bovine serum albumin column for the sample clean-up, followed by an achiral phenyl column (15.0×0.46cmI.D.) or a chiral amylose tris(3,5-dimethylphenylcarbamate) column (15.0×0.46cmI.D.). The chiral 2D HPLC method was applied to the development of a compromise condition for the in vitro metabolism of ABZ by means of experimental design involving multivariate analysis. Copyright © 2013 Elsevier B.V. All rights reserved.

In silico study of in vitro GPCR assays by QSAR modeling ...

EPA Pesticide Factsheets

The U.S. EPA is screening thousands of chemicals of environmental interest in hundreds of in vitro high-throughput screening (HTS) assays (the ToxCast program). One goal is to prioritize chemicals for more detailed analyses based on activity in molecular initiating events (MIE) of adverse outcome pathways (AOPs). However, the chemical space of interest for environmental exposure is much wider than this set of chemicals. Thus, there is a need to fill data gaps with in silico methods, and quantitative structure-activity relationships (QSARs) are a proven and cost effective approach to predict biological activity. ToxCast in turn provides relatively large datasets that are ideal for training and testing QSAR models. The overall goal of the study described here was to develop QSAR models to fill the data gaps in a larger environmental database of ~32k structures. The specific aim of the current work was to build QSAR models for 18 G-Protein Coupled Receptor (GPCR) assays, part of the aminergic category. Two QSAR modeling strategies were adopted: classification models were developed to separate chemicals into active/non-active classes, and then regression models were built to predict the potency values of the bioassays for the active chemicals. Multiple software programs were used to calculate constitutional, topological and substructural molecular descriptors from two-dimensional (2D) chemical structures. Model-fitting methods included PLSDA (partial least squares d

Antacid effects of Chinese herbal prescriptions assessed by a modified artificial stomach model

PubMed Central

Wu, Tsung-Hsiu; Chen, I-Chin; Chen, Lih-Chi

2010-01-01

AIM: To assess the antacid effects of the tonic Chinese herbal prescriptions, Si-Jun-Zi-Tang (SJZT) and Shen-Ling-Bai-Zhu-San (SLBZS). METHODS: Decoctions of the tonic Chinese herbal prescriptions, SJZT and SLBZS, were prepared according to Chinese original documents. The pH of the prescription decoctions and their neutralizing effects on artificial gastric acids were determined and compared with water and the active controls, sodium bicarbonate and colloidal aluminum phosphate. A modified model of Vatier’s artificial stomach was used to determine the duration of consistent neutralization effect on artificial gastric acids. The neutralization capacity in vitro was determined with the titration method of Fordtran’s model. RESULTS: The results showed that both SJZT and SLBZS have antacid effects in vitro. Compared with the water group, SJZT and SLBZS were found to possess significant gastric acid neutralizing effects. The duration for consistent neutralization of SLBZS was significantly longer than that of water. Also, SLBZS and SJZT exhibited significant antacid capacities compared to water. CONCLUSION: SJZT and SLBZS were consistently active in the artificial stomach model and are suggested to have antacid effects similar to the active control drugs. PMID:20845514

A new in vitro lipid digestion - in vivo absorption model to evaluate the mechanisms of drug absorption from lipid-based formulations.

PubMed

Crum, Matthew F; Trevaskis, Natalie L; Williams, Hywel D; Pouton, Colin W; Porter, Christopher J H

2016-04-01

In vitro lipid digestion models are commonly used to screen lipid-based formulations (LBF), but in vitro-in vivo correlations are in some cases unsuccessful. Here we enhance the scope of the lipid digestion test by incorporating an absorption 'sink' into the experimental model. An in vitro model of lipid digestion was coupled directly to a single pass in situ intestinal perfusion experiment in an anaesthetised rat. The model allowed simultaneous real-time analysis of the digestion and absorption of LBFs of fenofibrate and was employed to evaluate the influence of formulation digestion, supersaturation and precipitation on drug absorption. Formulations containing higher quantities of co-solvent and surfactant resulted in higher supersaturation and more rapid drug precipitation in vitro when compared to those containing higher quantities of lipid. In contrast, when the same formulations were examined using the coupled in vitro lipid digestion - in vivo absorption model, drug flux into the mesenteric vein was similar regardless of in vitro formulation performance. For some drugs, simple in vitro lipid digestion models may underestimate the potential for absorption from LBFs. Consistent with recent in vivo studies, drug absorption for rapidly absorbed drugs such as fenofibrate may occur even when drug precipitation is apparent during in vitro digestion.

Micropatterned coculture of hepatocytes on electrospun fibers as a potential in vitro model for predictive drug metabolism.

PubMed

Liu, Yaowen; Wei, Jiaojun; Lu, Jinfu; Lei, Dongmei; Yan, Shili; Li, Xiaohong

2016-06-01

The liver is the major organ of importance to determine drug dispositions in the body, thus the development of hepatocyte culture systems is of great scientific and practical interests to provide reliable and predictable models for in vitro drug screening. In the current study, to address the challenges of a rapid function loss of primary hepatocytes, the coculture of hepatocytes with fibroblasts and endothelial cells (Hep-Fib-EC) was established on micropatterned fibrous scaffolds. Liver-specific functions, such as the albumin secretion and urea synthesis, were well maintained in the coculture system, accompanied by a rapid formation of multicellular hepatocyte spheroids. The activities of phase I (CYP3A11 and CYP2C9) and phase II enzymes indicated a gradual increase for cocultured hepatocytes, and a maximum level was achieved after 5 days and maintained throughout 15 days of culture. The metabolism testing on model drugs indicated that the scaled clearance rates for hepatocytes in the Hep-Fib-EC coculture system were significantly higher than those of other culture methods, and a linear regression analysis indicated good correlations between the observed data of rats and in vitro predicted values during 15 days of culture. In addition, the enzyme activities and drug clearance rates of hepatocytes in the Hep-Fib-EC coculture model experienced sensitive responsiveness to the inducers and inhibitors of metabolizing enzymes. These results demonstrated the feasibility of micropatterned coculture of hepatocytes as a potential in vitro testing model for the prediction of in vivo drug metabolism. Copyright © 2016 Elsevier B.V. All rights reserved.

Development and Validation of a Computational Model for Androgen Receptor Activity

EPA Science Inventory

Testing thousands of chemicals to identify potential androgen receptor (AR) agonists or antagonists would cost millions of dollars and take decades to complete using current validated methods. High-throughput in vitro screening (HTS) and computational toxicology approaches can mo...

A catch-up validation study of an in vitro skin irritation test method using reconstructed human epidermis LabCyte EPI-MODEL24.

PubMed

Kojima, Hajime; Katoh, Masakazu; Shinoda, Shinsuke; Hagiwara, Saori; Suzuki, Tamie; Izumi, Runa; Yamaguchi, Yoshihiro; Nakamura, Maki; Kasahawa, Toshihiko; Shibai, Aya

2014-07-01

Three validation studies were conducted by the Japanese Society for Alternatives to Animal Experiments in order to assess the performance of a skin irritation assay using reconstructed human epidermis (RhE) LabCyte EPI-MODEL24 (LabCyte EPI-MODEL24 SIT) developed by the Japan Tissue Engineering Co., Ltd. (J-TEC), and the results of these studies were submitted to the Organisation for Economic Co-operation and Development (OECD) for the creation of a Test Guideline (TG). In the summary review report from the OECD, the peer review panel indicated the need to resolve an issue regarding the misclassification of 1-bromohexane. To this end, a rinsing operation intended to remove exposed chemicals was reviewed and the standard operating procedure (SOP) revised by J-TEC. Thereafter, in order to confirm general versatility of the revised SOP, a new validation management team was organized by the Japanese Center for the Validation of Alternative Methods (JaCVAM) to undertake a catch-up validation study that would compare the revised assay with similar in vitro skin irritation assays, per OECD TG No. 439 (2010). The catch-up validation and supplementary studies for LabCyte EPI-MODEL24 SIT using the revised SOPs were conducted at three laboratories. These results showed that the revised SOP of LabCyte EPI-MODEL24 SIT conformed more accurately to the classifications for skin irritation under the United Nations Globally Harmonised System of Classification and Labelling of Chemicals (UN GHS), thereby highlighting the importance of an optimized rinsing operation for the removal of exposed chemicals in obtaining consistent results from in vitro skin irritation assays. Copyright © 2013 John Wiley & Sons, Ltd.

Evaluating the Toxicity of Cigarette Whole Smoke Solutions in an Air-Liquid-Interface Human In Vitro Airway Tissue Model.

PubMed

Cao, Xuefei; Muskhelishvili, Levan; Latendresse, John; Richter, Patricia; Heflich, Robert H

2017-03-01

Exposure to cigarette smoke causes a multitude of pathological changes leading to tissue damage and disease. Quantifying such changes in highly differentiated in vitro human tissue models may assist in evaluating the toxicity of tobacco products. In this methods development study, well-differentiated human air-liquid-interface (ALI) in vitro airway tissue models were used to assess toxicological endpoints relevant to tobacco smoke exposure. Whole mainstream smoke solutions (WSSs) were prepared from 2 commercial cigarettes (R60 and S60) that differ in smoke constituents when machine-smoked under International Organization for Standardization conditions. The airway tissue models were exposed apically to WSSs 4-h per day for 1-5 days. Cytotoxicity, tissue barrier integrity, oxidative stress, mucin secretion, and matrix metalloproteinase (MMP) excretion were measured. The treatments were not cytotoxic and had marginal effects on tissue barrier properties; however, other endpoints responded in time- and dose-dependent manners, with the R60 resulting in higher levels of response than the S60 for many endpoints. Based on the lowest effect dose, differences in response to the WSSs were observed for mucin induction and MMP secretion. Mitigation of mucin induction by cotreatment of cultures with N-acetylcysteine suggests that oxidative stress contributes to mucus hypersecretion. Overall, these preliminary results suggest that quantifying disease-relevant endpoints using ALI airway models is a potential tool for tobacco product toxicity evaluation. Additional research using tobacco samples generated under smoking machine conditions that more closely approximate human smoking patterns will inform further methods development. Published by Oxford University Press on behalf of the Society of Toxicology 2017. This work is written by US Government employees and is in the public domain in the US.

Measurement of soil lead bioavailability and influence of soil types and properties: A review.

PubMed

Yan, Kaihong; Dong, Zhaomin; Wijayawardena, M A Ayanka; Liu, Yanju; Naidu, Ravi; Semple, Kirk

2017-10-01

Lead (Pb) is a widespread heavy metal which is harmful to human health, especially to young children. To provide a human health risk assessment that is more relevant to real conditions, Pb bioavailability in soils is increasingly employed in the assessment procedure. Both in vivo and in vitro measurements for lead bioavailability are available. In vivo models are time- consuming and expensive, while in vitro models are rapid, economic, reproducible, and reliable while involving more uncertainties. Uncertainties in various measurements create difficulties in accurately predicting Pb bioavailability, resulting in the unnecessary remediation of sites. In this critical review, we utilised available data from in vivo and in vitro studies to identify the key parameters influencing the in vitro measurements, and presented uncertainties existing in Pb bioavailability measurements. Soil type, properties and metal content are reported to influence lead bioavailability; however, the differences in methods for assessing bioavailability and the differences in Pb source limit one's ability to conduct statistical analyses on influences of soil factors on Pb bioavailability. The information provided in the review is fundamentally useful for the measurement of bioavailability and risk assessment practices. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dexamethasone diffusion across contact lenses is inhibited by Staphylococcus epidermidis biofilms in vitro

PubMed Central

Brothers, Kimberly M.; Nau, Amy C.; Romanowski, Eric G.; Shanks, Robert M. Q.

2014-01-01

Purpose This study was designed to measure the impact of bacterial biofilms on diffusion of an ocular therapeutic through silicone hydrogel bandage lenses in vitro. Methods An assay was designed to study the passage of a commonly used steroid dexamethasone through the silicone hydrogel soft contact lenses. Diffused dexamethasone was measured using a spectrophotometer over a period of 18 hours and quantified using a standard curve. This assay was performed with control and Staphylococcus epidermidis biofilm-coated contact lenses composed of lotrafilcon A and methafilcon. Biofilms were formed in brain heart infusion broth supplemented with D-glucose. Results The presented data validate a simple in vitro model that can be used to measure penetration of a topical therapeutic through silicone hydrogel soft contact lenses. Using this model we measured a reduction of dexamethasone diffusion by up to 88% through S. epidermidis biofilm-coated silicon hydrogel lenses compared to control lenses. Conclusions The results of this in vitro study demonstrate that bacterial biofilms impede dexamethasone diffusion through silicon hydrogel contact lenses, and warrant future studies regarding the clinical benefit of using ocular therapeutics in the setting of bandage contact lens use for corneal epithelial defects. PMID:25090165

Growing Three-Dimensional Corneal Tissue in a Bioreactor

NASA Technical Reports Server (NTRS)

Spaulding, Glen F.; Goodwin, Thomas J.; Aten, Laurie; Prewett, Tacey; Fitzgerald, Wendy S.; OConnor, Kim; Caldwell, Delmar; Francis, Karen M.

2003-01-01

Spheroids of corneal tissue about 5 mm in diameter have been grown in a bioreactor from an in vitro culture of primary rabbit corneal cells to illustrate the production of optic cells from aggregates and tissue. In comparison with corneal tissues previously grown in vitro by other techniques, this tissue approximates intact corneal tissue more closely in both size and structure. This novel three-dimensional tissue can be used to model cell structures and functions in normal and abnormal corneas. Efforts continue to refine the present in vitro method into one for producing human corneal tissue to overcome the chronic shortage of donors for corneal transplants: The method would be used to prepare corneal tissues, either from in vitro cultures of a patient s own cells or from a well-defined culture from another human donor known to be healthy. As explained in several articles in prior issues of NASA Tech Briefs, generally cylindrical horizontal rotating bioreactors have been developed to provide nutrient-solution environments conducive to the 30 NASA Tech Briefs, October 2003 growth of delicate animal cells, with gentle, low-shear flow conditions that keep the cells in suspension without damaging them. The horizontal rotating bioreactor used in this method, denoted by the acronym "HARV," was described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662), NASA Tech Briefs, Vol. 16, No. 5 (May, 1992), page 150.

In Vitro Interactions between Non-Steroidal Anti-Inflammatory Drugs and Antifungal Agents against Planktonic and Biofilm Forms of Trichosporon asahii

PubMed Central

Cong, Lin; Lu, Xuelian

2016-01-01

Increasing drug resistance has brought enormous challenges to the management of Trichosporon spp. infections. The in vitro antifungal activities of non-steroidal anti-inflammatory drugs (NSAIDs) against Candida spp. and Cryptococcus spp. were recently discovered. In the present study, the in vitro interactions between three NSAIDs (aspirin, ibuprofen and diclofenac sodium) and commonly used antifungal agents (fluconazole, itraconazole, voriconazole, caspofungin and amphotericin B) against planktonic and biofilm cells of T. asahii were evaluated using the checkerboard microdilution method. The spectrophotometric method and the XTT reduction assay were used to generate data on biofilm cells. The fractional inhibitory concentration index (FICI) and the ΔE model were compared to interpret drug interactions. Using the FICI, the highest percentages of synergistic effects against planktonic cells (86.67%) and biofilm cells (73.33%) were found for amphotericin B/ibuprofen, and caspofungin/ibuprofen showed appreciable percentages (73.33% for planktonic form and 60.00% for biofilm) as well. We did not observe antagonism. The ΔE model gave consistent results with FICI (86.67%). Our findings suggest that amphotericin B/ibuprofen and caspofungin/ibuprofen combinations have potential effects against T. asahii. Further in vivo and animal studies to investigate associated mechanisms need to be conducted. PMID:27275608

Screening Applications to Test Cellular Fitness in Transwell® Models After Nanoparticle Treatment.

PubMed

Christ, Bastian; Fey, Christina; Cubukova, Alevtina; Walles, Heike; Dembski, Sofia; Metzger, Marco

2017-01-01

Nanoparticles (NPs) in biotechnology hold great promise for revolutionizing medical treatments and therapies. In order to bring NPs into clinical application there is a number of preclinical in vitro and in vivo tests, which have to be applied before. The initial in vitro evaluation includes a detailed physicochemical characterization as well as biocompatibility tests, among others. For determination of biocompatibility at the cellular level, the correct choice of the in vitro assay as well as NP pretreatment is absolutely essential. There are a variety of assay technologies available that use standard plate readers to measure metabolic markers to estimate the number of viable cells in culture. Each cell viability assay has its own set of advantages and disadvantages. Regardless of the assay method chosen, the major factors critical for reproducibility and success include: (1) choosing the right assay after comparing optical NP properties with the read-out method of the assay, (2) verifying colloidal stability of NPs in cell culture media, (3) preparing a sterile and stable NP dispersion in cell culture media used in the assay, (4) using a tightly controlled and consistent cell model allowing appropriate characterization of NPs. This chapter will briefly summarize these different critical points, which can occur during biocompatibility screening applications of NPs.

Microengineering methods for cell-based microarrays and high-throughput drug-screening applications.

PubMed

Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

2011-09-01

Screening for effective therapeutic agents from millions of drug candidates is costly, time consuming, and often faces concerns due to the extensive use of animals. To improve cost effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems has facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell-based drug-screening models which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell-based drug-screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds great potential to provide repeatable 3D cell-based constructs with high temporal, spatial control and versatility.

A parameter estimation technique for stochastic self-assembly systems and its application to human papillomavirus self-assembly.

PubMed

Kumar, M Senthil; Schwartz, Russell

2010-12-09

Virus capsid assembly has been a key model system for studies of complex self-assembly but it does pose some significant challenges for modeling studies. One important limitation is the difficulty of determining accurate rate parameters. The large size and rapid assembly of typical viruses make it infeasible to directly measure coat protein binding rates or deduce them from the relatively indirect experimental measures available. In this work, we develop a computational strategy to deduce coat-coat binding rate parameters for viral capsid assembly systems by fitting stochastic simulation trajectories to experimental measures of assembly progress. Our method combines quadratic response surface and quasi-gradient descent approximations to deal with the high computational cost of simulations, stochastic noise in simulation trajectories and limitations of the available experimental data. The approach is demonstrated on a light scattering trajectory for a human papillomavirus (HPV) in vitro assembly system, showing that the method can provide rate parameters that produce accurate curve fits and are in good concordance with prior analysis of the data. These fits provide an insight into potential assembly mechanisms of the in vitro system and give a basis for exploring how these mechanisms might vary between in vitro and in vivo assembly conditions.

[Study on the Chinese herbal formula for treatment of vaginitis and the antimicrobial activity in murine models].

PubMed

Fu, Ting-ting; Wu, Jian-yuan; Wang, Li; Ma, Yao; Wang, Ying; Liu, Ying; Ding, Hong

2006-09-01

To study on the various proportions of Radix Sophorae Flavescentis, Cortex Phellodendri, Fructus Cnidii and pericarp of Zanthoxylum bungeanum Maxim in the formulas, whose antimicrobial effects on E. coli, S. aureus, P. aeruginosa and C. albicans under different pH values were compared in vitro. According to Chinese ancient proved recipe, the K-B method and plate diluting method were adopted to measure antimicrobial activity, and orthogonal design to ascertain the herbal formula in vitro. Finally, murine models were established to test the antimicrobial activity in vivo through vaginal membrane irritancy experiment, negative rate of pathogeny and pathological grade of vaginal membrane. The results suggested that formulas with different proportions of the herbs had diverse antimicrobial activities, and the effect was shown to be most obvious when one milliliter drug contains 100 microl Fructus Cnidii-pericarp of Zanthoxylum bungeanum (2:1) co-extracted volatile oil and 50 microl Radix Sophorae Flavescentis and Cortex Phenodendri ethanol extraction respectively under pH6. The antimicrobial effect of the formula, which hardly had any membrane irritancy, was better than Jie Eryin in vitro and vivo. The fromula has few components and better effect, and adaptation to the pH value of vaginitis. It is a promising alternative for gynecological diseases.

A parameter estimation technique for stochastic self-assembly systems and its application to human papillomavirus self-assembly

NASA Astrophysics Data System (ADS)

Senthil Kumar, M.; Schwartz, Russell

2010-12-01

Virus capsid assembly has been a key model system for studies of complex self-assembly but it does pose some significant challenges for modeling studies. One important limitation is the difficulty of determining accurate rate parameters. The large size and rapid assembly of typical viruses make it infeasible to directly measure coat protein binding rates or deduce them from the relatively indirect experimental measures available. In this work, we develop a computational strategy to deduce coat-coat binding rate parameters for viral capsid assembly systems by fitting stochastic simulation trajectories to experimental measures of assembly progress. Our method combines quadratic response surface and quasi-gradient descent approximations to deal with the high computational cost of simulations, stochastic noise in simulation trajectories and limitations of the available experimental data. The approach is demonstrated on a light scattering trajectory for a human papillomavirus (HPV) in vitro assembly system, showing that the method can provide rate parameters that produce accurate curve fits and are in good concordance with prior analysis of the data. These fits provide an insight into potential assembly mechanisms of the in vitro system and give a basis for exploring how these mechanisms might vary between in vitro and in vivo assembly conditions.

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

PubMed

Regdon, G; Vágó, I; Mándi, E; Regdon, G; Erós, I

2000-04-01

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

Microengineering Methods for Cell Based Microarrays and High-Throughput Drug Screening Applications

PubMed Central

Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

2011-01-01

Screening for effective therapeutic agents from millions of drug candidates is costly, time-consuming and often face ethical concerns due to extensive use of animals. To improve cost-effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems have facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell based drug-screening models, which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell based drug screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds a great potential to provide repeatable 3D cell based constructs with high temporal, spatial control and versatility. PMID:21725152

Patient-derived Models of Human Breast Cancer: Protocols for In vitro and In vivo Applications in Tumor Biology and Translational Medicine

PubMed Central

DeRose, Yoko S.; Gligorich, Keith M.; Wang, Guoying; Georgelas, Ann; Bowman, Paulette; Courdy, Samir J.; Welm, Alana L.; Welm, Bryan E.

2013-01-01

Research models that replicate the diverse genetic and molecular landscape of breast cancer are critical for developing the next generation therapeutic entities that can target specific cancer subtypes. Patient-derived tumorgrafts, generated by transplanting primary human tumor samples into immune-compromised mice, are a valuable method to model the clinical diversity of breast cancer in mice, and are a potential resource in personalized medicine. Primary tumorgrafts also enable in vivo testing of therapeutics and make possible the use of patient cancer tissue for in vitro screens. Described in this unit are a variety of protocols including tissue collection, biospecimen tracking, tissue processing, transplantation, and 3-dimensional culturing of xenografted tissue, that enable use of bona fide uncultured human tissue in designing and validating cancer therapies. PMID:23456611

Three-dimensional in vitro cancer spheroid models for Photodynamic Therapy: Strengths and Opportunities

NASA Astrophysics Data System (ADS)

Evans, Conor

2015-03-01

Three dimensional, in vitro spheroid cultures offer considerable utility for the development and testing of anticancer photodynamic therapy regimens. More complex than monolayer cultures, three-dimensional spheroid systems replicate many of the important cell-cell and cell-matrix interactions that modulate treatment response in vivo. Simple enough to be grown by the thousands and small enough to be optically interrogated, spheroid cultures lend themselves to high-content and high-throughput imaging approaches. These advantages have enabled studies investigating photosensitizer uptake, spatiotemporal patterns of therapeutic response, alterations in oxygen diffusion and consumption during therapy, and the exploration of mechanisms that underlie therapeutic synergy. The use of quantitative imaging methods, in particular, has accelerated the pace of three-dimensional in vitro photodynamic therapy studies, enabling the rapid compilation of multiple treatment response parameters in a single experiment. Improvements in model cultures, the creation of new molecular probes of cell state and function, and innovations in imaging toolkits will be important for the advancement of spheroid culture systems for future photodynamic therapy studies.

The novel kinase inhibitor ponatinib is an effective anti-angiogenic agent against neuroblastoma.

PubMed

Whittle, Sarah B; Patel, Kalyani; Zhang, Linna; Woodfield, Sarah E; Du, Michael; Smith, Valeria; Zage, Peter E

2016-12-01

Background High-risk neuroblastoma has poor outcomes with high rates of relapse despite aggressive treatment, and novel therapies are needed to improve these outcomes. Ponatinib is a multi-tyrosine kinase inhibitor that targets many pathways implicated in neuroblastoma pathogenesis. We hypothesized that ponatinib would be effective against neuroblastoma in preclinical models. Methods We evaluated the effects of ponatinib on survival and migration of human neuroblastoma cells in vitro. Using orthotopic xenograft mouse models of human neuroblastoma, we analyzed tumors treated with ponatinib for growth, gross and histologic appearance, and vascularity. Results Ponatinib treatment of neuroblastoma cells resulted in decreased cell viability and migration in vitro. In mice with orthotopic xenograft neuroblastoma tumors, treatment with ponatinib resulted in decreased growth and vascularity. Conclusions Ponatinib reduces neuroblastoma cell viability in vitro and reduces tumor growth and vascularity in vivo. The antitumor effects of ponatinib suggest its potential as a novel therapeutic agent for neuroblastoma, and further preclinical testing is warranted.

Comparison of manual and homogenizer methods for preparation of tick-derived stabilates of Theileria parva: equivalence testing using an in vitro titration model.

PubMed

Mbao, V; Speybroeck, N; Berkvens, D; Dolan, T; Dorny, P; Madder, M; Mulumba, M; Duchateau, L; Brandt, J; Marcotty, T

2005-07-01

Theileria parva sporozoite stabilates are used in the infection and treatment method of immunization, a widely accepted control option for East Coast fever in cattle. T. parva sporozoites are extracted from infected adult Rhipicephalus appendiculatus ticks either manually, using a pestle and a mortar, or by use of an electric homogenizer. A comparison of the two methods as a function of stabilate infectivity has never been documented. This study was designed to provide a quantitative comparison of stabilates produced by the two methods. The approach was to prepare batches of stabilate by both methods and then subject them to in vitro titration. Equivalence testing was then performed on the average effective doses (ED). The ratio of infective sporozoites yielded by the two methods was found to be 1.14 in favour of the manually ground stabilate with an upper limit of the 95% confidence interval equal to 1.3. We conclude that the choice of method rests more on costs, available infrastructure and standardization than on which method produces a richer sporozoite stabilate.

Radical scavenging activities of Rio Red grapefruits and Sour orange fruit extracts in different in vitro model systems.

PubMed

Jayaprakasha, G K; Girennavar, Basavaraj; Patil, Bhimanagouda S

2008-07-01

Antioxidant fractions from two different citrus species such as Rio Red (Citrus paradise Macf.) and Sour orange (Citrus aurantium L.) were extracted with five different polar solvents using Soxhlet type extractor. The total phenolic content of the extracts was determined by Folin-Ciocalteu method. Ethyl acetate extract of Rio Red and Sour orange was found to contain maximum phenolics. The dried fractions were screened for their antioxidant activity potential using in vitro model systems such as 1,1-diphenyl-2-picryl hydrazyl (DPPH), phosphomolybdenum method and nitroblue tetrazolium (NBT) reduction at different concentrations. The methanol:water (80:20) fraction of Rio Red showed the highest radical scavenging activity 42.5%, 77.8% and 92.1% at 250, 500 and 1000 ppm, respectively, while methanol:water (80:20) fraction of Sour orange showed the lowest radical scavenging activity at all the tested concentrations. All citrus fractions showed good antioxidant capacity by the formation of phosphomolybdenum complex at 200 ppm. In addition, superoxide radical scavenging activity was assayed using non-enzymatic (NADH/phenaxine methosulfate) superoxide generating system. All the extracts showed variable superoxide radical scavenging activity. Moreover, methanol:water (80:20) extract of Rio Red and methanol extract of Sour orange exhibited marked reducing power in potassium ferricyanide reduction method. The data obtained using above in vitro models clearly establish the antioxidant potential of citrus fruit extracts. However, comprehensive studies need to be conducted to ascertain the in vivo bioavailability, safety and efficacy of such extracts in experimental animals. To the best of our knowledge, this is the first report on antioxidant activity of different polar extracts from Rio Red and Sour oranges.

In situ electrochemical assessment of cytotoxicity of chlorophenols in MCF-7 and HeLa cells.

PubMed

Qin, Hongwei; Liu, Jiguang; Zhang, Zeshi; Li, Jinlian; Gao, Guanggang; Yang, Yuxin; Yuan, Xing; Wu, Dongmei

2014-10-01

An in situ electrochemical method was used to assess the cytotoxicity of chlorophenols using human breast cancer (MCF-7) and cervical carcinoma (HeLa) cells as models. On treatment with different chlorophenols, the electrochemical responses of the selected cells, resulting from the oxidation of guanine and xanthine in the cytoplasm, indicated the cell viability. In addition, the in situ in vitro electrochemical method was further compared with the traditional MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Although similar cytotoxicity data were obtained from both methods, the effective concentrations of chlorophenols that inhibited 50% cell growth (EC50 values) from the electrochemical method were only slightly lower than those from the MTT assay. These results indicate that the in situ in vitro electrochemical method paves a simple, rapid, strongly responsive, and label-free way to the cytotoxicity assessment of different chlorophenol pollutants. Copyright © 2014 Elsevier Inc. All rights reserved.

Comparison of the occlusal contact area of virtual models and actual models: a comparative in vitro study on Class I and Class II malocclusion models.

PubMed

Lee, Hyemin; Cha, Jooly; Chun, Youn-Sic; Kim, Minji

2018-06-19

The occlusal registration of virtual models taken by intraoral scanners sometimes shows patterns which seem much different from the patients' occlusion. Therefore, this study aims to evaluate the accuracy of virtual occlusion by comparing virtual occlusal contact area with actual occlusal contact area using a plaster model in vitro. Plaster dental models, 24 sets of Class I models and 20 sets of Class II models, were divided into a Molar, Premolar, and Anterior group. The occlusal contact areas calculated by the Prescale method and the virtual occlusion by scanning method were compared, and the ratio of the molar and incisor area were compared in order to find any particular tendencies. There was no significant difference between the Prescale results and the scanner results in both the molar and premolar groups (p = 0.083 and 0.053, respectively). On the other hand, there was a significant difference between the Prescale and the scanner results in the anterior group with the scanner results presenting overestimation of the occlusal contact points (p < 0.05). In Molars group, the regression analysis shows that the two variables express linear correlation and has a linear equation with a slope of 0.917. R 2 is 0.930. Groups of Premolars and Anteriors had a week linear relationship and greater dispersion. Difference between the actual and virtual occlusion revealed in the anterior portion, where overestimation was observed in the virtual model obtained from the scanning method. Nevertheless, molar and premolar areas showed relatively accurate occlusal contact area in the virtual model.

Development of a convenient in vitro fertilization method using interspecific hybrids between Oryzias latipes and Oryzias curvinotus.

PubMed

Kamei, Yasuhiro; Itou, Junji; Oda, Shoji; Masui, Mami; Kim, Jin-Hyeong; Ishikawa, Tomoko; Yuba, Shunsuke; Kinoshita, Masato; Mitani, Hiroshi; Todo, Takeshi

2007-12-01

Medaka is a small Asian freshwater teleost and has been an excellent model for fertilization studies for more than 50 years. Therefore, experimental procedures for in vitro fertilization (IVF) and cryopreservation of sperm are well established. In contrast, since the eggs or early embryos can not be cryopreserved, many females are killed to obtain unfertilized eggs for IVF. Recent progress in genomics is establishing medaka as a new model animal in functional genomics, and numerous mutant and transgenic strains have been established and stored as frozen sperm. Accumulated preserved resources require a simple and reliable recovery method for IVF. In this paper, we describe a method for obtaining a large number of unfertilized eggs without killing females, using sterile interspecific hybrids between Oryzias latipes and O. curvinotus. However, there is no report about the normality of offspring that were obtained by IVF using unfertilized eggs spawned in mating with the sterile hybrid male. In this paper, we have confirmed the reliability of the method regarding the influences on the next generation and also assessed conditions for efficient collection of unfertilized eggs. The method would be useful not only for fertilization studies but also for keeping transgenics and mutants, including a mutant library for a reverse genetic approach.

76 FR 4113 - Independent Scientific Peer Review Panel Meeting on an In Vitro

Federal Register 2010, 2011, 2012, 2013, 2014

2011-01-24

... Vitro Estrogen Receptor Transcriptional Activation Test Method for Endocrine Disruptor Chemical... Vitro Estrogen Receptor Transcriptional Activation Test Method for Endocrine Disruptor Chemical... the information included in the BRD supports ICCVAM's draft test method recommendations. NICEATM...

Correlation Between An In-vitro Method And An In-vivo Method In Assessing Bioavailable Arsenic In Two Pesticide-Amended Soils

NASA Astrophysics Data System (ADS)

Quazi, S.; Sarkar, D.; Sylvia, V.; Datta, R.

2006-05-01

Health risk assessment of Arsenic (As) enriched soil requires the estimation of bioavailable fraction of total metal. Research has been conducted to gain a better understanding of the relationship between metal availability and risk assessment. Some baseline risk assessments developed for contaminated sites have used the conservative assumption that all (i.e. 100%) of the As present in soils and wastes is bioavailable, due to tremendous cost associated with in-vivo bioavailability studies. This potentially overestimates the actual health risk, elevating the expenses associated with site cleanup. Health risk from direct exposure to soil-As via the hand-to-mouth exposure route is restricted only to those fractions of As in the soil that are available to the human gastrointestinal system. A reasonable approach is to develop in-vitro methods that simulate the complex and dynamic human gastrointestinal system and correlate well with the results of in-vivo method. Thus this study aims in addressing the potential of one such in-vitro method developed by our research group in assessing the bioavailability of soil-As. Two soils with drastically different chemical characteristics in regards to As reactivity (Immokalee-low As retention capacity; Millhopper-high As retention capacity) spiked with a pesticide (sodium arsenate) were used. Soils were amended at two rates representing concentrations typically found at Superfund sites: 675 and 1500 mg/kg of As. In-vitro bioavailability experiments were performed in order to obtain an estimate of the amount of As likely to be available in the human gastrointestinal system as well as the fraction potentially absorbed onto the intestinal linings. Following the in-vitro study selective in-vivo bioavailability studies using As-contaminated soils were conducted on male and female mice to validate the in-vitro results via comparison with the in-vivo data. Soils were administered orally to the BALB/c mice immediately after spiking. Treatments comprised of a soil group (As in soil), a positive control group (only As) and a negative control group (no soil, no As). Blood samples were collected at different time periods to determine As concentrations. Correlation between the in-vitro and in-vivo data was determined. Information obtained from this study will serve as the first step towards the future development of a semi-quantitative model for predicting bioavailable As. This in turn will result in designing appropriate, cost-effective remedial strategies for As contaminated sites. Keywords: Bioavailability, In-vitro, In-vivo, Arsenic, Soil, Risk Assessment

A study of the uptake and biodistribution of nano-titanium dioxide using in vitro and in vivo models of oral intake

NASA Astrophysics Data System (ADS)

MacNicoll, Alan; Kelly, Mick; Aksoy, Hatice; Kramer, Evelien; Bouwmeester, Hans; Chaudhry, Qasim

2015-02-01

Certain food additives may contain a sizeable fraction of particles in the nanoscale. However, little is known about the fate, behaviour and toxicological effects of orally-ingested nanoparticles. This study investigated the uptake and biodistribution of nano- and larger-sized titanium dioxide (TiO2) using an in vitro model of gut epithelium and in vivo in rat. The results of the in vivo study showed that oral administration of 5 mg/kg body weight of TiO2 nano- or larger particles did not lead to any significant translocation of TiO2 (measured as titanium) either to blood, urine or to various organs in rat at any of the time intervals studied over a 96 h post-administration period. Different methods used for dispersing particles did not affect the uptake, and orally administered TiO2 was found excreted in the faeces over a period of time. The in vitro study provided further evidence for the lack of translocation of TiO2 across the gut epithelium model. The overall evidence from both in vivo and in vitro studies did not support that oral ingestion of nano- or larger particles of TiO2 via food would result in any significant internal exposure of the consumer to the nanoparticles. The dietary TiO2 nanoparticles are likely to be excreted in the faeces.

Therapeutic potential of Pirfenidone for treating equine corneal scarring

PubMed Central

Fink, Michael K.; Giuliano, Elizabeth A.; Tandon, Ashish; Mohan, Rajiv R.

2014-01-01

Objective To evaluate the safety and efficacy of Pirfenidone (PFD) in the treatment of equine corneal fibrosis using an in vitro model. Methods Healthy donor equine corneas were collected and used to generate primary equine corneal fibroblasts (ECFs) by growing cultures in minimal essential medium supplemented with 10% fetal bovine serum. Equine corneal myofibroblasts (ECMs), used as a model of equine corneal fibrosis, were produced by growing ECF cultures in serum-free medium containing transforming growth factor β1 (1ng/ml). Trypan blue viability assays and changes in ECF morphology were utilized to determine the optimal PFD dose for this in vitro model. Trypan blue viability, phase contrast microscopy, and TUNEL assays were used to evaluate the cytotoxicity of PFD. Scratch and MTT assays were used to evaluate the effect of PFD on cellular migration and proliferation. Real-time PCR, immunoblot analysis, and immunocytochemistry were employed to determine the efficacy of PFD to inhibit ECM formation in vitro. Results Topical PFD application at 200 μg/ml successfully decreased αSMA expression when compared to the TGFβ1 only treatment group (P < 0.01). PFD application ≤ 200 μg/ml did not affect ECF phenotype or cellular viability and did not result in significant cytotoxicity. Conclusions Pirfenidone safely and effectively inhibits TGFβ1-induced equine corneal fibrosis in vitro. In vivo studies are warranted. PMID:25041235

Insulin catalyzes the curcumin-induced wound healing: An in vitro model for gingival repair

PubMed Central

Singh, Neetu; Ranjan, Vishal; Zaidi, Deeba; Shyam, Hari; Singh, Aparna; Lodha, Divya; Sharma, Ramesh; Verma, Umesh; Dixit, Jaya; Balapure, Anil K.

2012-01-01

Objectives: Human gingival fibroblasts (hGFs) play a major role in the maintenance and repair of gingival connective tissue. The mitogen insulin with IGFs etc. synergizes in facilitating wound repair. Although curcumin (CUR) and insulin regulate apoptosis, their impact as a combination on hGF in wound repair remains unknown. Our study consists of: 1) analysis of insulin-mediated mitogenesis on CUR-treated hGF cells, and 2) development of an in vitro model of wound healing. Materials and Methods: Apoptotic rate in CUR-treated hGF cells with and without insulin was observed by AnnexinV/PI staining, nuclear morphological analysis, FACS and DNA fragmentation studies. Using hGF confluent cultures, wounds were mechanically created in vitro and incubated with the ligands for 48 h in 0.2% fetal bovine serum DMEM. Results: CUR alone showed dose-dependent (1–50 μM) effects on hGF. Insulin (1 μg/ml) supplementation substantially enhanced cell survival through up-regulation of mitogenesis/anti-apoptotic elements. Conclusions: The in vitro model for gingival wound healing establishes that insulin significantly enhanced wound filling faster than CUR-treated hGF cells over 48 h. This reinforces the pivotal role of insulin in supporting CUR-mediated wound repair. The findings have significant bearing in metabolic dysfunctions, e.g. diabetes, atherosclerosis, etc., especially under Indian situations. PMID:23087505

An ex vivo approach to botanical-drug interactions: a proof of concept study.

PubMed

Wang, Xinwen; Zhu, Hao-Jie; Munoz, Juliana; Gurley, Bill J; Markowitz, John S

2015-04-02

Botanical medicines are frequently used in combination with therapeutic drugs, imposing a risk for harmful botanical-drug interactions (BDIs). Among the existing BDI evaluation methods, clinical studies are the most desirable, but due to their expense and protracted time-line for completion, conventional in vitro methodologies remain the most frequently used BDI assessment tools. However, many predictions generated from in vitro studies are inconsistent with clinical findings. Accordingly, the present study aimed to develop a novel ex vivo approach for BDI assessment and expand the safety evaluation methodology in applied ethnopharmacological research. This approach differs from conventional in vitro methods in that rather than botanical extracts or individual phytochemicals being prepared in artificial buffers, human plasma/serum collected from a limited number of subjects administered botanical supplements was utilized to assess BDIs. To validate the methodology, human plasma/serum samples collected from healthy subjects administered either milk thistle or goldenseal extracts were utilized in incubation studies to determine their potential inhibitory effects on CYP2C9 and CYP3A4/5, respectively. Silybin A and B, two principal milk thistle phytochemicals, and hydrastine and berberine, the purported active constituents in goldenseal, were evaluated in both phosphate buffer and human plasma based in vitro incubation systems. Ex vivo study results were consistent with formal clinical study findings for the effect of milk thistle on the disposition of tolbutamide, a CYP2C9 substrate, and for goldenseal׳s influence on the pharmacokinetics of midazolam, a widely accepted CYP3A4/5 substrate. Compared to conventional in vitro BDI methodologies of assessment, the introduction of human plasma into the in vitro study model changed the observed inhibitory effect of silybin A, silybin B and hydrastine and berberine on CYP2C9 and CYP3A4/5, respectively, results which more closely mirrored those generated in clinical study. Data from conventional buffer-based in vitro studies were less predictive than the ex vivo assessments. Thus, this novel ex vivo approach may be more effective at predicting clinically relevant BDIs than conventional in vitro methods. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

An in vitro model of Mycobacterium leprae induced granuloma formation

PubMed Central

2013-01-01

Background Leprosy is a contagious and chronic systemic granulomatous disease caused by Mycobacterium leprae. In the pathogenesis of leprosy, granulomas play a key role, however, the mechanisms of the formation and maintenance of M. leprae granulomas are still not clearly understood. Methods To better understand the molecular physiology of M. leprae granulomas and the interaction between the bacilli and human host cells, we developed an in vitro model of human granulomas, which mimicked the in vivo granulomas of leprosy. Macrophages were differentiated from human monocytes, and infected with M. leprae, and then cultured with autologous human peripheral blood mononuclear cells (PBMCs). Results Robust granuloma-like aggregates were obtained only when the M. leprae infected macrophages were co-cultured with PBMCs. Histological examination showed M. leprae within the cytoplasmic center of the multinucleated giant cells, and these bacilli were metabolically active. Macrophages of both M1 and M2 types co-existed in the granuloma like aggregates. There was a strong relationship between the formation of granulomas and changes in the expression levels of cell surface antigens on macrophages, cytokine production and the macrophage polarization. The viability of M. leprae isolated from granulomas indicated that the formation of host cell aggregates benefited the host, but the bacilli also remained metabolically active. Conclusions A simple in vitro model of human M. leprae granulomas was established using human monocyte-derived macrophages and PBMCs. This system may be useful to unravel the mechanisms of disease progression, and subsequently develop methods to control leprosy. PMID:23782413

SkinEthic Laboratories, a company devoted to develop and produce in vitro alternative methods to animal use.

PubMed

de Brugerolle, Anne

2007-01-01

SkinEthic Laboratories is a France-based biotechnology company recognised as the world leader in tissue engineering. SkinEthic is devoted to develop and produce reliable and robust in vitro alternative methods to animal use in cosmetic, chemical and pharmaceutical industries. SkinEthic models provide relevant tools for efficacy and safety screening tests in order to support an integrated decision-making during research and development phases. Some screening tests are referenced and validated as alternatives to animal use (Episkin), others are in the process of validation under ECVAM and OECD guidelines. SkinEthic laboratories provide a unique and joined experience of more than 20 years from Episkin SNC and SkinEthic SA. Their unique cell culture process allows in vitro reconstructed human tissues with well characterized histology, functionality and ultrastructure features to be mass produced. Our product line includes skin models: a reconstructed human epidermis with a collagen layer, Episkin, reconstructed human epidermis without or with melanocytes (with a tanning degree from phototype II to VI) and a reconstructed human epithelium, i.e. cornea, and other mucosa, i.e. oral, gingival, oesophageal and vaginal. Our philosophy is based on 3 main commitments: to support our customers by providing robust and reliable models, to ensure training and education in using validated protocols, allowing a large array of raw materials, active ingredients and finished products in solid, liquid, powder, cream or gel form to be screened, and, to provide a dedicated service to our partners.

Immune Cell-Supplemented Human Skin Model for Studying Fungal Infections.

PubMed

Kühbacher, Andreas; Sohn, Kai; Burger-Kentischer, Anke; Rupp, Steffen

2017-01-01

Human skin is a niche for various fungal species which either colonize the surface of this tissue as commensals or, primarily under conditions of immunosuppression, invade the skin and cause infection. Here we present a method for generation of a human in vitro skin model supplemented with immune cells of choice. This model represents a complex yet amenable tool to study molecular mechanisms of host-fungi interactions at human skin.

In-vitro development of vitrified-warmed bovine oocytes after activation may be predicted based on mathematical modelling of cooling and warming rates during vitrification, storage and sample removal.

PubMed

Sansinena, Marina; Santos, Maria Victoria; Chirife, Jorge; Zaritzky, Noemi

2018-05-01

Heat transfer during cooling and warming is difficult to measure in cryo-devices; mathematical modelling is an alternative method that can describe these processes. In this study, we tested the validity of one such model by assessing in-vitro development of vitrified and warmed bovine oocytes after parthenogenetic activation and culture. The viability of oocytes vitrified in four different cryo-devices was assessed. Consistent with modelling predictions, oocytes vitrified using cryo-devices with the highest modelled cooling rates had significantly (P < 0.05) better cleavage and blastocyst formation rates. We then evaluated a two-step sample removal process, in which oocytes were held in nitrogen vapour for 15 s to simulate sample identification during clinical application, before being removed completely and warmed. Oocytes exposed to this procedure showed reduced developmental potential, according to the model, owing to thermodynamic instability and devitrification at relatively low temperatures. These findings suggest that cryo-device selection and handling, including method of removal from nitrogen storage, are critical to survival of vitrified oocytes. Limitations of the study include use of parthenogenetically activated rather than fertilized ova and lack of physical measurement of recrystallization. We suggest mathematical modelling could be used to predict the effect of critical steps in cryopreservation. Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Bioaccessibility and digestive stability of carotenoids in cooked eggs studied using a dynamic in vitro gastrointestinal model.

PubMed

Nimalaratne, Chamila; Savard, Patricia; Gauthier, Sylvie F; Schieber, Andreas; Wu, Jianping

2015-03-25

Among dietary carotenoids, lutein and zeaxanthin are known to protect against age-related macular degeneration, a leading cause of irreversible vision loss in the elderly. Egg yolk is rich in lutein and zeaxanthin, however, the effect of cooking and gastrointestinal digestion on yolk carotenoids is poorly understood. An in vitro dynamic gastrointestinal model (TIM-1) was used to investigate the digestive stability and bioaccessibility of carotenoids from boiled, fried, and scrambled eggs. Bioaccessibility but not digestive stability was significantly affected by the method of cooking. The main egg carotenoids, all-E-lutein and all-E-zeaxanthin, were stable during the digestion with average recoveries of 90 and 88%, respectively. No trans-cis isomerization of carotenoids was observed during digestion. Both all-E-lutein and all-E-zeaxanthin from scrambled eggs showed significantly lower bioaccessibility compared to boiled eggs. The results indicate that the bioaccessibility of egg carotenoids can be affected by different food preparation methods.

Characterization and isolation of highly purified porcine satellite cells

PubMed Central

Ding, Shijie; Wang, Fei; Liu, Yan; Li, Sheng; Zhou, Guanghong; Hu, Ping

2017-01-01

Pig is an important food source and an excellent system to model human diseases. Careful characterization of the swine skeletal muscle stem cells (satellite cells) will shed lights on generation of swine skeletal muscle disease model and efficient production of porcine meat for the food industry. Paired box protein 7 (Pax7) is a highly conserved transcription factor shared by satellite cells from various species. However, the sequence of Pax7 has not been characterized in pig. The lack of method to isolate highly purified satellite cells hinders the thorough characterization of the swine satellite cells. Here we found molecular markers for swine satellite cells and revealed that the porcine satellite cells were heterogeneous in various pieces of skeletal muscle. We further developed a method to isolate highly purified satellite cells directly from porcine muscles using fluorescence-activated cell sorting. We next characterized the proliferation and differentiation abilities of isolated satellite cells in vitro; and found that long-term culturing of satellite cells in vitro led to stemness loss. PMID:28417015

Visualisation of distribution of gold nanoparticles in liver tissues ex vivo and in vitro using the method of optical coherence tomography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Genina, Elina A; Terentyuk, G S; Khlebtsov, B N

2012-06-30

The possibility of visualising the distribution of gold nanoparticles in liver by means of the method of optical coherence tomography is studied experimentally in model samples of beef liver in vitro and rat liver ex vivo. In the experiments we used the gold nanoparticles in the form of nanocages with resonance absorption in the near-IR spectral region. In the model studies the suspension of nanoparticles was applied to the surface of the sample, which then was treated with ultrasound. In the ex vivo studies the suspension of nanoparticles was injected to the laboratory rats intravenously. The image contrast and themore » optical depth of detection of blood vessels and liver structure components are calculated, as well as the depth of liver optical probing before and after the injection of nanoparticles. It was shown that the administration of the nanoparticle increases significantly the imaging contrast of liver blood vessels owing to the localisation of the nanoparticles therein.« less

Photoreceptor Outer Segment-like Structures in Long-Term 3D Retinas from Human Pluripotent Stem Cells.

PubMed

Wahlin, Karl J; Maruotti, Julien A; Sripathi, Srinivasa R; Ball, John; Angueyra, Juan M; Kim, Catherine; Grebe, Rhonda; Li, Wei; Jones, Bryan W; Zack, Donald J

2017-04-10

The retinal degenerative diseases, which together constitute a leading cause of hereditary blindness worldwide, are largely untreatable. Development of reliable methods to culture complex retinal tissues from human pluripotent stem cells (hPSCs) could offer a means to study human retinal development, provide a platform to investigate the mechanisms of retinal degeneration and screen for neuroprotective compounds, and provide the basis for cell-based therapeutic strategies. In this study, we describe an in vitro method by which hPSCs can be differentiated into 3D retinas with at least some important features reminiscent of a mature retina, including exuberant outgrowth of outer segment-like structures and synaptic ribbons, photoreceptor neurotransmitter expression, and membrane conductances and synaptic vesicle release properties consistent with possible photoreceptor synaptic function. The advanced outer segment-like structures reported here support the notion that 3D retina cups could serve as a model for studying mature photoreceptor development and allow for more robust modeling of retinal degenerative disease in vitro.

Reliability of In Vitro Methods used to Measure Intrinsic Clearance of Hydrophobic Organic Chemicals by Rainbow Trout: Results of an International Ring Trial.

PubMed

Nichols, John; Fay, Kellie; Bernhard, Mary Jo; Bischof, Ina; Davis, John; Halder, Marlies; Hu, Jing; Johanning, Karla; Laue, Heike; Nabb, Diane; Schlechtriem, Christian; Segner, Helmut; Swintek, Joe; Weeks, John; Embry, Michelle

2018-05-14

In vitro assays are widely employed to obtain intrinsic clearance estimates used in toxicokinetic modeling efforts. However, the reliability of these methods is seldom reported. Here we describe the results of an international ring trial designed to evaluate two in vitro assays used to measure intrinsic clearance in rainbow trout. An important application of these assays is to predict the effect of biotransformation on chemical bioaccumulation. Six laboratories performed substrate depletion experiments with cyclohexyl salicylate, fenthion, 4-n-nonylphenol, deltamethrin, methoxychlor, and pyrene using cryopreserved hepatocytes and liver S9 fractions from trout. Variability within and among laboratories was characterized as the percent coefficient of variation (CV) in measured in vitro intrinsic clearance rates (CLIN VITRO, INT; ml/h/mg protein or 106 cells) for each chemical and test system. Mean intra-laboratory CVs for each test chemical averaged 18.9% for hepatocytes and 14.1% for S9 fractions, while inter-laboratory CVs (all chemicals and all tests) averaged 30.1% for hepatocytes and 22.4% for S9 fractions. When CLIN VITRO, INT values were extrapolated to in vivo intrinsic clearance estimates (CLIN VIVO,INT; L/d/kg fish), both assays yielded similar levels of activity (< 4-fold difference for all chemicals). Hepatic clearance rates (CLH; L/d/kg fish) calculated using data from both assays exhibited even better agreement. These findings show that both assays are highly reliable and suggest that either may be used to inform chemical bioaccumulation assessments for fish. This study highlights several issues related to the demonstration of assay reliability and may provide a template for evaluating other in vitro biotransformation assays.

pH-cycling models for in vitro evaluation of the efficacy of fluoridated dentifrices for caries control: strengths and limitations

PubMed Central

BUZALAF, Marília Afonso Rabelo; HANNAS, Angélica Reis; MAGALHÃES, Ana Carolina; RIOS, Daniela; HONÓRIO, Heitor Marques; DELBEM, Alberto Carlos Botazzo

2010-01-01

Despite a plethora of in situ studies and clinical trials evaluating the efficacy of fluoridated dentifrices on caries control, in vitro pH cycling models are still broadly used because they mimic the dynamics of mineral loss and gain involved in caries formation. This paper critically reviews the current literature on existing pH-cycling models for the in vitro evaluation of the efficacy of fluoridated dentifrices for caries control, focusing on their strengths and limitations. A search was undertaken in the MEDLINE electronic journal database using the keywords "pH-cycling", "demineralization", "remineralization", "in vitro", "fluoride", "dentifrice". The primary outcome was the decrease of demineralization or the increase of remineralization as measured by different methods (e.g.: transverse microradiography) or tooth fluoride uptake. Inclusion of studies, data extraction and quality assessment were undertaken independently and in duplicate by two members of the review team. Disagreements were solved by discussion and consensus or by a third party. One hundred and sixteen studies were included, of which 42 addressed specifically the comparison of dentifrices using different pH-cycling models. The other studies included meta-analysis or reviews, data about the effect of different fluoride sources on de-remineralization, different methods for analysis de-remineralization and chemical variables and characteristics of dental hard tissues that might have influence on de-remineralization processes. Generally, the studies presented ability to detect known results established by clinical trials, to demonstrate dose-related responses in the fluoride content of the dentifrices, and to provide repeatability and reproducibility between tests. In order to accomplish these features satisfactorily, it is mandatory to take into account the type of substrate and baseline artificial lesion, as well as the adequate response variables and statistical approaches to be used. This critical review of literature showed that the currently available pH-cycling models are appropriate to detect dose-response and pH-response of fluoride dentifrices, and to evaluate the impact of new active principles on the effect of fluoridated dentifrices, as well as their association with other anti-caries treatments. PMID:20835565

Stenotrophomonas maltophilia: emergence of multidrug-resistant strains during therapy and in an in vitro pharmacodynamic chamber model.

PubMed Central

Garrison, M W; Anderson, D E; Campbell, D M; Carroll, K C; Malone, C L; Anderson, J D; Hollis, R J; Pfaller, M A

1996-01-01

Emergence of Stenotrophomonas maltophilia as a nosocomial pathogen is becoming increasingly apparent. Pleiotropic resistance characterizes S. maltophilia. Furthermore, a slow growth rate and an increased mutation rate generate discordance between in vitro susceptibility testing and clinical outcome. Despite original susceptibility, drug-resistant strains of S. maltophilia are often recovered from patients receiving beta-lactams, quinolones, or aminoglycosides. Given the disparity among various in vitro susceptibility methods, this study incorporated a unique pharmacodynamic model to more accurately characterize the bacterial time-kill curves and mutation rates of four clinical isolates of S. maltophilia following exposure to simulated multidose regimens of ceftazidime, ciprofloxacin, gentamicin, and ticarcillin-clavulanate. Time-kill data demonstrated regrowth of S. maltophilia with all four agents. With the exception of ticarcillin-clavulanate, viable bacterial counts at the end of 24 h exceeded the starting inoculum. Ciprofloxacin only reduced bacterial counts by less than 1.0 log prior to rapid bacterial regrowth. Resistant mutant strains, identical to their parent strain by pulsed-field gel electrophoresis, were observed following exposure to each class of antibiotic. Mutant strains also had distinct susceptibility patterns. These data are consistent with previous reports which suggest that S. maltophilia, despite susceptibility data that imply that the organism is sensitive, develops multiple forms of resistance quickly and against several classes of antimicrobial agents. Standard in vitro susceptibility methods are not completely reliable for detecting resistant S. maltophilia strains; and therefore, interpretation of these results should be done with caution. In vivo studies are needed to determine optimal therapy against S. maltophilia infections. PMID:9124855

Good cell culture practices &in vitro toxicology.

PubMed

Eskes, Chantra; Boström, Ann-Charlotte; Bowe, Gerhard; Coecke, Sandra; Hartung, Thomas; Hendriks, Giel; Pamies, David; Piton, Alain; Rovida, Costanza

2017-12-01

Good Cell Culture Practices (GCCP) is of high relevance to in vitro toxicology. The European Society of Toxicology In Vitro (ESTIV), the Center for Alternatives for Animal Testing (CAAT) and the In Vitro Toxicology Industrial Platform (IVTIP) joined forces to address by means of an ESTIV 2016 pre-congress session the different aspects and applications of GCCP. The covered aspects comprised the current status of the OECD guidance document on Good In Vitro Method Practices, the importance of quality assurance for new technological advances in in vitro toxicology including stem cells, and the optimized implementation of Good Manufacturing Practices and Good Laboratory Practices for regulatory testing purposes. General discussions raised the duality related to the difficulties in implementing GCCP in an academic innovative research framework on one hand, and on the other hand, the need for such GCCP principles in order to ensure reproducibility and robustness of in vitro test methods for toxicity testing. Indeed, if good cell culture principles are critical to take into consideration for all uses of in vitro test methods for toxicity testing, the level of application of such principles may depend on the stage of development of the test method as well as on the applications of the test methods, i.e., academic innovative research vs. regulatory standardized test method. Copyright © 2017 Elsevier Ltd. All rights reserved.

Combined Biochemical, Biophysical, and Cellular Methods to Study Fe-S Cluster Transfer and Cytosolic Aconitase Repair by MitoNEET.

PubMed

Mons, Cécile; Ferecatu, Ioana; Riquier, Sylvie; Lescop, Ewen; Bouton, Cécile; Golinelli-Cohen, Marie-Pierre

2017-01-01

MitoNEET is the first identified Fe-S protein anchored to mammalian outer mitochondrial membranes with the vast majority of the protein polypeptide located in the cytosol, including its [2Fe-2S] cluster-binding domain. The coordination of the cluster is unusual and involves three cysteines and one histidine. MitoNEET is capable of transferring its redox-active Fe-S cluster to a bacterial apo-ferredoxin in vitro even under aerobic conditions, unlike other Fe-S transfer proteins such as ISCU. This specificity suggests its possible involvement in Fe-S repair after oxidative and/or nitrosative stress. Recently, we identified cytosolic aconitase/iron regulatory protein 1 (IRP1) as the first physiological protein acceptor of the mitoNEET Fe-S cluster in an Fe-S repair process. This chapter describes methods to study in vitro mitoNEET Fe-S cluster transfer/repair to a bacterial ferredoxin used as a model aporeceptor and in a more comprehensive manner to cytosolic aconitase/IRP1 after a nitrosative stress using in vitro, in cellulo, and in vivo methods. © 2017 Elsevier Inc. All rights reserved.

Validation of Alternative In Vitro Methods to Animal Testing: Concepts, Challenges, Processes and Tools.

PubMed

Griesinger, Claudius; Desprez, Bertrand; Coecke, Sandra; Casey, Warren; Zuang, Valérie

This chapter explores the concepts, processes, tools and challenges relating to the validation of alternative methods for toxicity and safety testing. In general terms, validation is the process of assessing the appropriateness and usefulness of a tool for its intended purpose. Validation is routinely used in various contexts in science, technology, the manufacturing and services sectors. It serves to assess the fitness-for-purpose of devices, systems, software up to entire methodologies. In the area of toxicity testing, validation plays an indispensable role: "alternative approaches" are increasingly replacing animal models as predictive tools and it needs to be demonstrated that these novel methods are fit for purpose. Alternative approaches include in vitro test methods, non-testing approaches such as predictive computer models up to entire testing and assessment strategies composed of method suites, data sources and decision-aiding tools. Data generated with alternative approaches are ultimately used for decision-making on public health and the protection of the environment. It is therefore essential that the underlying methods and methodologies are thoroughly characterised, assessed and transparently documented through validation studies involving impartial actors. Importantly, validation serves as a filter to ensure that only test methods able to produce data that help to address legislative requirements (e.g. EU's REACH legislation) are accepted as official testing tools and, owing to the globalisation of markets, recognised on international level (e.g. through inclusion in OECD test guidelines). Since validation creates a credible and transparent evidence base on test methods, it provides a quality stamp, supporting companies developing and marketing alternative methods and creating considerable business opportunities. Validation of alternative methods is conducted through scientific studies assessing two key hypotheses, reliability and relevance of the test method for a given purpose. Relevance encapsulates the scientific basis of the test method, its capacity to predict adverse effects in the "target system" (i.e. human health or the environment) as well as its applicability for the intended purpose. In this chapter we focus on the validation of non-animal in vitro alternative testing methods and review the concepts, challenges, processes and tools fundamental to the validation of in vitro methods intended for hazard testing of chemicals. We explore major challenges and peculiarities of validation in this area. Based on the notion that validation per se is a scientific endeavour that needs to adhere to key scientific principles, namely objectivity and appropriate choice of methodology, we examine basic aspects of study design and management, and provide illustrations of statistical approaches to describe predictive performance of validated test methods as well as their reliability.

In vitro dynamic model simulating the digestive tract of 6-month-old infants.

PubMed

Passannanti, Francesca; Nigro, Federica; Gallo, Marianna; Tornatore, Fabio; Frasso, Annalisa; Saccone, Giulia; Budelli, Andrea; Barone, Maria V; Nigro, Roberto

2017-01-01

In vivo assays cannot always be conducted because of ethical reasons, technical constraints or costs, but a better understanding of the digestive process, especially in infants, could be of great help in preventing food-related pathologies and in developing new formulas with health benefits. In this context, in vitro dynamic systems to simulate human digestion and, in particular, infant digestion could become increasingly valuable. To simulate the digestive process through the use of a dynamic model of the infant gastroenteric apparatus to study the digestibility of starch-based infant foods. Using M.I.D.A (Model of an Infant Digestive Apparatus), the oral, gastric and intestinal digestibility of two starch-based products were measured: 1) rice starch mixed with distilled water and treated using two different sterilization methods (the classical method with a holding temperature of 121°C for 37 min and the HTST method with a holding temperature of 137°C for 70 sec) and 2) a rice cream with (premium product) or without (basic product) an aliquot of rice flour fermented by Lactobacillus paracasei CBA L74. After the digestion the foods were analyzed for the starch concentration, the amount of D-glucose released and the percentage of hydrolyzed starch. An in vitro dynamic system, which was referred to as M.I.D.A., was obtained. Using this system, the starch digestion occurred only during the oral and intestinal phase, as expected. The D-glucose released during the intestinal phase was different between the classical and HTST methods (0.795 grams for the HTST versus 0.512 for the classical product). The same analysis was performed for the basic and premium products. In this case, the premium product had a significant difference in terms of the starch hydrolysis percentage during the entire process. The M.I.D.A. system was able to digest simple starches and a more complex food in the correct compartments. In this study, better digestibility of the premium product was revealed.

Alternative prediction methods of protein and energy evaluation of pig feeds.

PubMed

Święch, Ewa

2017-01-01

Precise knowledge of the actual nutritional value of individual feedstuffs and complete diets for pigs is important for efficient livestock production. Methods of assessment of protein and energy values in pig feeds have been briefly described. In vivo determination of protein and energy values of feeds in pigs are time-consuming, expensive and very often require the use of surgically-modified animals. There is a need for more simple, rapid, inexpensive and reproducible methods for routine feed evaluation. Protein and energy values of pig feeds can be estimated using the following alternative methods: 1) prediction equations based on chemical composition; 2) animal models as rats, cockerels and growing pigs for adult animals; 3) rapid methods, such as the mobile nylon bag technique and in vitro methods. Alternative methods developed for predicting the total tract and ileal digestibility of nutrients including amino acids in feedstuffs and diets for pigs have been reviewed. This article focuses on two in vitro methods that can be used for the routine evaluation of amino acid ileal digestibility and energy value of pig feeds and on factors affecting digestibility determined in vivo in pigs and by alternative methods. Validation of alternative methods has been carried out by comparing the results obtained using these methods with those acquired in vivo in pigs. In conclusion, energy and protein values of pig feeds may be estimated with satisfactory precision in rats and by the two- or three-step in vitro methods providing equations for the calculation of standardized ileal digestibility of amino acids and metabolizable energy content. The use of alternative methods of feed evaluation is an important way for reduction of stressful animal experiments.

Kinetic analysis of hyperpolarized data with minimum a priori knowledge: Hybrid maximum entropy and nonlinear least squares method (MEM/NLS).

PubMed

Mariotti, Erika; Veronese, Mattia; Dunn, Joel T; Southworth, Richard; Eykyn, Thomas R

2015-06-01

To assess the feasibility of using a hybrid Maximum-Entropy/Nonlinear Least Squares (MEM/NLS) method for analyzing the kinetics of hyperpolarized dynamic data with minimum a priori knowledge. A continuous distribution of rates obtained through the Laplace inversion of the data is used as a constraint on the NLS fitting to derive a discrete spectrum of rates. Performance of the MEM/NLS algorithm was assessed through Monte Carlo simulations and validated by fitting the longitudinal relaxation time curves of hyperpolarized [1-(13) C] pyruvate acquired at 9.4 Tesla and at three different flip angles. The method was further used to assess the kinetics of hyperpolarized pyruvate-lactate exchange acquired in vitro in whole blood and to re-analyze the previously published in vitro reaction of hyperpolarized (15) N choline with choline kinase. The MEM/NLS method was found to be adequate for the kinetic characterization of hyperpolarized in vitro time-series. Additional insights were obtained from experimental data in blood as well as from previously published (15) N choline experimental data. The proposed method informs on the compartmental model that best approximate the biological system observed using hyperpolarized (13) C MR especially when the metabolic pathway assessed is complex or a new hyperpolarized probe is used. © 2014 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc.

Sex differences in the MB49 syngeneic, murine model of bladder cancer

PubMed Central

White-Gilbertson, Shai; Davis, Megan; Voelkel-Johnson, Christina; Kasman, Laura M.

2016-01-01

OBJECTIVE The MB49 syngeneic, murine model of bladder cancer has been widely used for more than 35 years. In humans, bladder cancer is one third as prevalent in women as in men, with a trend toward lower prevalence in parous compared to nulliparous women. Our objective was to determine if the MB49 bladder cancer model reproduces the sex differences observed in humans, and to determine its sensitivity to testosterone and the pregnancy hormone, human chorionic gonadotropin (hCG). METHODS Male and female C57BL/6 mice were implanted with MB49 murine bladder cancer cells, and observed for tumor growth. MB49 dose responses to hCG and dihydrotestosterone were determined in vitro. RESULTS MB49 tumor growth was significantly greater in male mice than female mice. Pregnancy did not affect MB49 tumor growth in female mice. MB49 cells did not proliferate in response to hCG in vitro and the functional receptor for gonadotropins was absent. Dihydrotestosterone strongly stimulated growth of MB49 cells in vitro. CONCLUSIONS The MB49 murine model of bladder cancer reproduced some aspects of the sex differences observed in humans. Our results suggest that testosterone may stimulate MB49 cell proliferation, which may explain the more rapid MB49 tumor growth observed in male mice. PMID:26998503

Suitability of [18F]altanserin and PET to determine 5-HT2A receptor availability in the rat brain: in vivo and in vitro validation of invasive and non-invasive kinetic models.

PubMed

Kroll, Tina; Elmenhorst, David; Matusch, Andreas; Wedekind, Franziska; Weisshaupt, Angela; Beer, Simone; Bauer, Andreas

2013-08-01

While the selective 5-hydroxytryptamine type 2a receptor (5-HT2AR) radiotracer [18F]altanserin is well established in humans, the present study evaluated its suitability for quantifying cerebral 5-HT2ARs with positron emission tomography (PET) in albino rats. Ten Sprague Dawley rats underwent 180 min PET scans with arterial blood sampling. Reference tissue methods were evaluated on the basis of invasive kinetic models with metabolite-corrected arterial input functions. In vivo 5-HT2AR quantification with PET was validated by in vitro autoradiographic saturation experiments in the same animals. Overall brain uptake of [18F]altanserin was reliably quantified by invasive and non-invasive models with the cerebellum as reference region shown by linear correlation of outcome parameters. Unlike in humans, no lipophilic metabolites occurred so that brain activity derived solely from parent compound. PET data correlated very well with in vitro autoradiographic data of the same animals. [18F]Altanserin PET is a reliable tool for in vivo quantification of 5-HT2AR availability in albino rats. Models based on both blood input and reference tissue describe radiotracer kinetics adequately. Low cerebral tracer uptake might, however, cause restrictions in experimental usage.

Rapid multislice T1 mapping of mouse myocardium: Application to quantification of manganese uptake in α-Dystrobrevin knockout mice.

PubMed

Jiang, Kai; Li, Wen; Li, Wei; Jiao, Sen; Castel, Laurie; Van Wagoner, David R; Yu, Xin

2015-11-01

The aim of this study was to develop a rapid, multislice cardiac T1 mapping method in mice and to apply the method to quantify manganese (Mn(2+)) uptake in a mouse model with altered Ca(2+) channel activity. An electrocardiography-triggered multislice saturation-recovery Look-Locker method was developed and validated both in vitro and in vivo. A two-dose study was performed to investigate the kinetics of T1 shortening, Mn(2+) relaxivity in myocardium, and the impact of Mn(2+) on cardiac function. The sensitivity of Mn(2+)-enhanced MRI in detecting subtle changes in altered Ca(2+) channel activity was evaluated in a mouse model with α-dystrobrevin knockout. Validation studies showed strong agreement between the current method and an established method. High Mn(2+) dose led to significantly accelerated T1 shortening. Heart rate decreased during Mn(2+) infusion, while ejection ratio increased slightly at the end of imaging protocol. No statistical difference in cardiac function was detected between the two dose groups. Mice with α-dystrobrevin knockout showed enhanced Mn(2+) uptake in vivo. In vitro patch-clamp study showed increased Ca(2+) channel activity. The saturation recovery method provides rapid T1 mapping in mouse hearts, which allowed sensitive detection of subtle changes in Mn(2+) uptake in α-dystrobrevin knockout mice. © 2014 Wiley Periodicals, Inc.

Prediction of percutaneous absorption in human using three-dimensional human cultured epidermis LabCyte EPI-MODEL.

PubMed

Hikima, Tomohiro; Kaneda, Noriaki; Matsuo, Kyouhei; Tojo, Kakuji

2012-01-01

The objective of this study is to establish a relationship of the skin penetration parameters between the three-dimensional cultured human epidermis LabCyte EPI-MODEL (LabCyte) and hairless mouse (HLM) skin penetration in vitro and to predict the skin penetration and plasma concentration profile in human. The skin penetration experiments through LabCyte and HLM skin were investigated using 19 drugs that have a different molecular weight and lipophilicity. The penetration flux for LabCyte reached 30 times larger at maximum than that for HLM skin. The human data can be estimated from the in silico approach with the diffusion coefficient (D), the partition coefficient (K) and the skin surface concentration (C) of drugs by assuming the bi-layer skin model for both LabCyte and HLM skin. The human skin penetration of β-estradiol, prednisolone, testosterone and ethynylestradiol was well agreed between the simulated profiles and in vitro experimental data. Plasma concentration profiles of β-estradiol in human were also simulated and well agreed with the clinical data. The present alternative method may decrease human or animal skin experiment for in vitro skin penetration.

β-cyclodextrin-poly(β-amino ester) nanoparticles for sustained drug delivery across the blood-brain barrier.

PubMed

Gil, Eun Seok; Wu, Linfeng; Xu, Lichong; Lowe, Tao Lu

2012-11-12

Novel biodegradable polymeric nanoparticles composed of β-cyclodextrin and poly(β-amino ester) segments have been developed for sustained drug delivery across the blood-brain barrier (BBB). The nanoparticles have been synthesized by cross-linking β-cyclodextrin with poly(β-amino ester) via the Michael addition method. The chemical, physical, and degradation properties of the nanoparticles have been characterized by matrix-assisted laser desoption/ionization time-of-flight, attenuated total reflectance Fourier transform infrared spectroscopy, nuclear magnetic resonance, dynamic light scattering, and atomic force microscopy techniques. Bovine and human brain microvascular endothelial cell monolayers have been constructed as in vitro BBB models. Preliminary results show that the nanoparticles do not affect the integrity of the in vitro BBB models, and the nanoparticles have much higher permeability than dextran control across the in vitro BBB models. Doxorubicin has been loaded into the nanoparticles with a loading efficiency of 86%, and can be released from the nanoparticles for at least one month. The developed β-cyclodextrin-poly(β-amino ester) nanoparticles might be useful as drug carriers for transporting drugs across the BBB to treat chronic diseases in the brain.

Development, current applications and future roles of biorelevant two-stage in vitro testing in drug development.

PubMed

Fiolka, Tom; Dressman, Jennifer

2018-03-01

Various types of two stage in vitro testing have been used in a number of experimental settings. In addition to its application in quality control and for regulatory purposes, two-stage in vitro testing has also been shown to be a valuable technique to evaluate the supersaturation and precipitation behavior of poorly soluble drugs during drug development. The so-called 'transfer model', which is an example of two-stage testing, has provided valuable information about the in vivo performance of poorly soluble, weakly basic drugs by simulating the gastrointestinal drug transit from the stomach into the small intestine with a peristaltic pump. The evolution of the transfer model has resulted in various modifications of the experimental model set-up. Concomitantly, various research groups have developed simplified approaches to two-stage testing to investigate the supersaturation and precipitation behavior of weakly basic drugs without the necessity of using a transfer pump. Given the diversity among the various two-stage test methods available today, a more harmonized approach needs to be taken to optimize the use of two stage testing at different stages of drug development. © 2018 Royal Pharmaceutical Society.

Anti-inflammatory, antiproliferative and cytoprotective potential of the Attalea phalerata Mart. ex Spreng. pulp oil

PubMed Central

Lescano, Caroline Honaiser; Arrigo, Jucicléia da Silva; Cardoso, Cláudia Andrea Lima; Coutinho, Janclei Pereira; Moslaves, Iluska Senna Bonfá; Ximenes, Thalita Vieira do Nascimento; Kadri, Monica Cristina Toffoli; Weber, Simone Schneider; Perdomo, Renata Trentin; Kassuya, Cândida Aparecida Leite; Vieira, Maria do Carmo; Sanjinez-Argandoña, Eliana Janet

2018-01-01

The anti-inflammatory, antiproliferative and cytoprotective activity of the Attalea phalerata Mart. ex Spreng pulp oil was evaluated by in vitro and in vivo methods. As for the chemical profile, the antioxidant activity was performed by spectrophotometry, and the profile of carotenoids and amino acids by chromatography. Our data demonstrated that A. phalerata oil has high carotenoid content, antioxidant activity and the presence of 5 essential amino acids. In the in vitro models of inflammation, the oil demonstrated the capacity to inhibit COX1 and COX2 enzymes, the production of nitric oxide and also induces macrophages to spreading. In the in vivo models of inflammation, the oil inhibited edema and leukocyte migration in the Wistar rats. In the in vitro model of antiproliferative and cytoprotective activity, the oil was shown inactive against the kidney carcinoma and prostate carcinoma lineage cells and with cytoprotective capacity in murine fibroblast cells, inhibiting the cytotoxic action of doxorubicin. Therefore, it is concluded that A. phalerata pulp oil has anti-inflammatory effects with nutraceutical properties potential due to the rich composition. Moreover, the oil also has cytoprotective activity probably because of its ability to inhibit the action of free radicals. PMID:29634766

20171015 - Integrating Toxicity, Toxicokinetic, and Exposure Data for Risk-based Chemical Alternatives Assessment (ISES)

EPA Science Inventory

In order to predict the margin between the dose needed for adverse chemical effects and actual human exposure rates, data on hazard, exposure, and toxicokinetics are needed. In vitro methods, biomonitoring, and mathematical modeling have provided initial estimates for many extant...

Screening-level assays for potentially human-infectious environmental Legionella spp.

EPA Science Inventory

In spite of the fact that Legionella species can be isolated from nonclinical settings, there is no standard method to determine whether environmental legionellae may be infectious to humans. In this study, an in vivo murine model of pneumonia and three in vitro proliferation as...

Human health and the environment: Predicting plasma protein binding and metabolic clearance rates of environmentally relevant chemicals.

EPA Science Inventory

In silico methods provide a rapid, inexpensive means of screening a wide array of environmentally relevant pollutants, pesticides, fungicides and consumer products for further toxicity testing. Physiologically based pharmacokinetic (PBPK) models bridge the gap between in vitro as...

Independent data validation of an in vitro method for ...

EPA Pesticide Factsheets

In vitro bioaccessibility assays (IVBA) estimate arsenic (As) relative bioavailability (RBA) in contaminated soils to improve the accuracy of site-specific human exposure assessments and risk calculations. For an IVBA assay to gain acceptance for use in risk assessment, it must be shown to reliably predict in vivo RBA that is determined in an established animal model. Previous studies correlating soil As IVBA with RBA have been limited by the use of few soil types as the source of As. Furthermore, the predictive value of As IVBA assays has not been validated using an independent set of As-contaminated soils. Therefore, the current study was undertaken to develop a robust linear model to predict As RBA in mice using an IVBA assay and to independently validate the predictive capability of this assay using a unique set of As-contaminated soils. Thirty-six As-contaminated soils varying in soil type, As contaminant source, and As concentration were included in this study, with 27 soils used for initial model development and nine soils used for independent model validation. The initial model reliably predicted As RBA values in the independent data set, with a mean As RBA prediction error of 5.3% (range 2.4 to 8.4%). Following validation, all 36 soils were used for final model development, resulting in a linear model with the equation: RBA = 0.59 * IVBA + 9.8 and R2 of 0.78. The in vivo-in vitro correlation and independent data validation presented here provide

An Alternative Method for Long-Term Culture of Chicken Embryonic Stem Cell In Vitro.

PubMed

Zhang, Li; Wu, Yenan; Li, Xiang; Wei, Shao; Xing, Yiming; Lian, Zhengxing; Han, Hongbing

2018-01-01

Chicken embryonic stem cells (cESCs) obtained from stage X embryos provide a novel model for the study of avian embryonic development. A new way to maintain cESCs for a long period in vitro still remains unexplored. We found that the cESCs showed stem cell-like properties in vitro for a long term with the support of DF-1 feeder and basic culture medium supplemented with human basic fibroblast growth factor (hbFGF), mouse stem cell factor (mSCF), and human leukemia inhibitory factor (hLIF). During the long culture period, the cESCs showed typical ES cell morphology and expressed primitive stem cell markers with a relatively stable proliferation rate and high telomerase activity. These cells also exhibited the capability to differentiate into cardiac myocytes, smooth muscle cells, neural cells, osteoblast, and adipocyte in vitro . Chimera chickens were produced by cESCs cultured for 25 passages with this new culture system. The experiments showed that DF-1 was the optimal feeder and hbFGF was an important factor for maintaining the pluripotency of cESCs in vitro .

The Production of 3D Tumor Spheroids for Cancer Drug Discovery

PubMed Central

Sant, Shilpa; Johnston, Paul A.

2017-01-01

New cancer drug approval rates are ≤ 5% despite significant investments in cancer research, drug discovery and development. One strategy to improve the rate of success of new cancer drugs transitioning into the clinic would be to more closely align the cellular models used in the early lead discovery with pre-clinical animal models and patient tumors. For solid tumors, this would mandate the development and implementation of three dimensional (3D) in vitro tumor models that more accurately recapitulate human solid tumor architecture and biology. Recent advances in tissue engineering and regenerative medicine have provided new techniques for 3D spheroid generation and a variety of in vitro 3D cancer models are being explored for cancer drug discovery. Although homogeneous assay methods and high content imaging approaches to assess tumor spheroid morphology, growth and viability have been developed, the implementation of 3D models in HTS remains challenging due to reasons that we discuss in this review. Perhaps the biggest obstacle to achieve acceptable HTS assay performance metrics occurs in 3D tumor models that produce spheroids with highly variable morphologies and/or sizes. We highlight two methods that produce uniform size-controlled 3D multicellular tumor spheroids that are compatible with cancer drug research and HTS; tumor spheroids formed in ultra-low attachment microplates, or in polyethylene glycol dimethacrylate hydrogel microwell arrays. PMID:28647083

In Vitro Interactions between Aspirin and Amphotericin B against Planktonic Cells and Biofilm Cells of Candida albicans and C. parapsilosis

PubMed Central

Zhou, Yabin; Wang, Ganggang; Li, Yutang; Liu, Yang; Song, Yu; Zheng, Wenshuai; Zhang, Ning; Hu, Xiaoyan; Yan, Shikun

2012-01-01

The increase in drug resistance and invasion caused by biofilm formation brings enormous challenges to the management of Candida infection. Aspirin's antibiofilm activity in vitro was discovered recently. The spectrophotometric method and the XTT {2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide} reduction assay used for data generation make it possible to evaluate fungal biofilm growth accurately. The combined use of the most commonly used methods, the fractional inhibitory concentration index (FICI) and a newly developed method, the ΔE model, which uses the concentration-effect relationship over the whole concentration range instead of using the MIC index alone, makes the interpretation of results more reliable. As an attractive tool for studying the pharmacodynamics of antimicrobial agents, time-kill curves can provide detailed information about antimicrobial efficacy as a function of both time and concentration. In the present study, in vitro interactions between aspirin (acetylsalicylic acid [ASA]) and amphotericin B (AMB) against planktonic cells and biofilm cells of Candida albicans and C. parapsilosis were evaluated by the checkerboard microdilution method and the time-kill test. Synergistic and indifferent effects were found for the combination of ASA and AMB against planktonic cells, while strong synergy was found against biofilm cells analyzed by FICI. The ΔE model gave more consistent results with FICI. The positive interactions in concentration were also confirmed by the time-kill test. Moreover, this approach also revealed the pharmacodynamics changes of ASA and synergistic action on time. Our findings suggest a potential clinical use for combination therapy with ASA and AMB to augment activity against biofilm-associated infections. PMID:22391539

Cellular High-Energy Cavitation Trauma - Description of a Novel In Vitro Trauma Model in Three Different Cell Types.

PubMed

Cao, Yuli; Risling, Mårten; Malm, Elisabeth; Sondén, Anders; Bolling, Magnus Frödin; Sköld, Mattias K

2016-01-01

The mechanisms involved in traumatic brain injury have yet to be fully characterized. One mechanism that, especially in high-energy trauma, could be of importance is cavitation. Cavitation can be described as a process of vaporization, bubble generation, and bubble implosion as a result of a decrease and subsequent increase in pressure. Cavitation as an injury mechanism is difficult to visualize and model due to its short duration and limited spatial distribution. One strategy to analyze the cellular response of cavitation is to employ suitable in vitro models. The flyer-plate model is an in vitro high-energy trauma model that includes cavitation as a trauma mechanism. A copper fragment is accelerated by means of a laser, hits the bottom of a cell culture well causing cavitation, and shock waves inside the well and cell medium. We have found the flyer-plate model to be efficient, reproducible, and easy to control. In this study, we have used the model to analyze the cellular response to microcavitation in SH-SY5Y neuroblastoma, Caco-2, and C6 glioma cell lines. Mitotic activity in neuroblastoma and glioma was investigated with BrdU staining, and cell numbers were calculated using automated time-lapse imaging. We found variations between cell types and between different zones surrounding the lesion with these methods. It was also shown that the injured cell cultures released S-100B in a dose-dependent manner. Using gene expression microarray, a number of gene families of potential interest were found to be strongly, but differently regulated in neuroblastoma and glioma at 24 h post trauma. The data from the gene expression arrays may be used to identify new candidates for biomarkers in cavitation trauma. We conclude that our model is useful for studies of trauma in vitro and that it could be applied in future treatment studies.

CON4EI: Evaluation of QSAR models for hazard identification and labelling of eye irritating chemicals.

PubMed

Geerts, L; Adriaens, E; Alépée, N; Guest, R; Willoughby, J A; Kandarova, H; Drzewiecka, A; Fochtman, P; Verstraelen, S; Van Rompay, A R

2018-06-01

Assessment of ocular irritation is a regulatory requirement in safety evaluation of industrial and consumer products. Although a number of in vitro ocular irritation assays exist, none are capable of fully categorizing chemicals as stand-alone assays. Therefore, the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was developed to assess the reliability of eight in vitro test methods and computational models as well as establishing an optimal tiered-testing strategy. For three computational models (Toxtree, and Case Ultra EYE_DRAIZE and EYE_IRR) performance parameters were calculated. Coverage ranged from 15 to 58%. Coverage was 2 to 3.4 times higher for liquids than for solids. The lowest number of false positives (5%) was reached with EYE_IRR; this model however also gave a high number of false negatives (46%). The lowest number of false negatives (25%) was seen with Toxtree; for liquids Toxtree predicted the lowest number of false negatives (11%), for solids EYE_DRAIZE did (17%). It can be concluded that the training sets should be enlarged with high quality data. The tested models are not yet sufficiently powerful for stand-alone evaluations, but that they can surely become of value in an integrated weight-of-evidence approach in hazard assessment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development and Application of a Polymicrobial in vitro Wound Biofilm Model

PubMed Central

Woods, Jeremy; Boegli, Laura; Kirker, Kelly R.; Agostinho, Alessandra M.; Durch, Amanda M.; Pulcini, Elinor deLancey; Stewart, Philip S.; James, Garth A.

2012-01-01

Aims The goal of this investigation was to develop an in vitro, polymicrobial, wound biofilm capable of supporting the growth of bacteria with variable oxygen requirements. Methods and Results The strict anaerobe Clostridium perfringens was isolated by cultivating wound homogenates using the drip-flow reactor, and a three-species biofilm model was established using methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and C. perfringens in the colony-drip-flow reactor model. Plate counts revealed that MRSA, P. aeruginosa, and C. perfringens grew to 7.39±0.45, 10.22±0.22, and 7.13±0.77 log CFU per membrane, respectively. The three-species model was employed to evaluate the efficacy of two antimicrobial dressings, Curity™ AMD and Acticoat™, compared to sterile gauze controls. Microbial growth on Curity™ AMD and gauze were not significantly different, for any species, whereas Acticoat™ was found to significantly reduce growth for all three species. Conclusions Using the Colony-DFR, a three-species biofilm was successfully grown, and the biofilms displayed a unique structure consisting of distinct layers that appeared to be inhabited exclusively or predominantly by a single species. Significance and Impact of Study The primary accomplishment of this study was the isolation and growth of an obligate anaerobe in an in vitro model without establishing an artificially anaerobic environment. PMID:22353049

A novel approach for in vitro meat production.

PubMed

Pandurangan, Muthuraman; Kim, Doo Hwan

2015-07-01

The present review describes the possibility of in vitro meat production with the help of advanced co-culturing methods. In vitro meat production method could be a possible alternative for the conventional meat production. Originally, the research on in vitro meat production was initiated by the National Aeronautics and Space Administration (NASA) for space voyages. The required key qualities for accepting in vitro meat for consumption would be good efficiency ratio, increased protein synthesis rate in skeletal muscles, and mimicking the conventional meat qualities. In vitro culturing of meat is possible with the use of skeletal muscle tissue engineering, stem cell, cell co-culture, and tissue culture methods. Co-culture of myoblast and fibroblast is believed as one of the major techniques for in vitro meat production. In our lab, we have co-cultured myoblast and fibroblast. We believe that a billion pounds of in vitro meat could be produced from one animal for consumption. However, we require a great deal of research on in vitro meat production.

Quantum dot labeling and tracking of cultured limbal epithelial cell transplants in-vitro

PubMed Central

Genicio, Nuria; Paramo, Juan Gallo; Shortt, Alex J.

2015-01-01

PURPOSE Cultured human limbal epithelial cells (HLEC) have shown promise in the treatment of limbal stem cell deficiency but little is known about their survival, behaviour and long-term fate post transplantation. The aim of this research was to evaluate, in-vitro, quantum dot (QDot) technology as a tool for tracking transplanted HLEC. METHODS In-vitro cultured HLEC were labeled with Qdot nanocrystals. Toxicity was assessed using live-dead assays. The effect on HLEC function was assessed using colony forming efficiency assays and expression of CK3, P63alpha and ABCG2. Sheets of cultured HLEC labeled with Qdot nanocrystals were transplanted onto decellularised human corneo-scleral rims in an organ culture model and observed to investigate the behaviour of transplanted cells. RESULTS Qdot labeling had no detrimental effect on HLEC viability or function in-vitro. Proliferation resulted in a gradual reduction in Qdot signal but sufficient signal was present to allow tracking of cells through multiple generations. Cells labeled with Qdots could be reliably detected and observed using confocal microscopy for at least 2 weeks post transplantation in our organ culture model. In addition it was possible to label and observe epithelial cells in intact human corneas using the Rostock corneal module adapted for use with the Heidelberg HRA. CONCLUSIONS This work demonstrates that Qdots combined with existing clinical equipment could be used to track HLEC for up to 2 weeks post transplantation, however, our model does not permit the assessment of cell labeling beyond 2 weeks. Further characterisation in in-vivo models are required. PMID:26024089

Human induced pluripotent stem cells can reach complete terminal maturation: in vivo and in vitro evidence in the erythropoietic differentiation model

PubMed Central

Kobari, Ladan; Yates, Frank; Oudrhiri, Noufissa; Francina, Alain; Kiger, Laurent; Mazurier, Christelle; Rouzbeh, Shaghayegh; El-Nemer, Wassim; Hebert, Nicolas; Giarratana, Marie-Catherine; François, Sabine; Chapel, Alain; Lapillonne, Hélène; Luton, Dominique; Bennaceur-Griscelli, Annelise; Douay, Luc

2012-01-01

Background Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors. Design and Methods We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathological induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation. Results We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch from fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathological induced pluripotent stem cells into mice. Conclusions These results demonstrate that human induced pluripotent stem cells: i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation; and ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment. PMID:22733021

Evaluation of in vitro models for predicting acidosis risk of barley grain in finishing beef cattle.

PubMed

Anele, U Y; Swift, M-L; McAllister, T A; Galyean, M L; Yang, W Z

2015-10-01

Our objective was to develop a model to predict the acidosis potential of barley based on the in vitro batch culture incubation of 50 samples varying in bulk density, starch content, processing method, growing location, and agronomic practices. The model was an adaptation of the acidosis index (calculated from a combination of in situ and in vitro analyses and from several components of grain chemical composition) developed in Australia for use in the feed industry to estimate the potential for grains to increase the risk of ruminal acidosis. Of the independent variables considered, DM disappearance at 6 h of incubation (DMD6) using reduced-strength (20%) buffer in the batch culture accounted for 90.5% of the variation in the acidosis index with a root mean square error (RMSE) of 4.46%. To evaluate our model using independent datasets (derived from previous batch culture studies using full-strength [100%] buffer), we performed another batch culture study using full-strength buffer. The full-strength buffer model using in vitro DMD6 (DMD6-FS) accounted for 66.5% of the variation in the acidosis index with an RMSE of 8.30%. When the new full-strength buffer model was applied to 3 independent datasets to predict acidosis, it accounted for 20.1, 28.5, and 30.2% of the variation in the calculated acidosis index. Significant ( < 0.001) mean bias was evident in 2 of the datasets, for which the DMD6 model underpredicted the acidosis index by 46.9 and 5.73%. Ranking of samples from the most diverse independent dataset using the DMD6-FS model and the Black (2008) model (calculated using in situ starch degradation) indicated the relationship between the rankings using Spearman's rank correlation was negative (ρ = -0.30; = 0.059). When the reduced-strength buffer model was used, however, there were similarities in the acidosis index ranking of barley samples by the models as shown by the result of a correlation analysis between calculated (using the Australian model) and predicted (using the reduced-strength buffer DMD6 model) acidosis index (ρ = 0.67; < 0.001). Results suggest that our model, which is based on a reduced-strength buffer in vitro DMD6, has the potential to predict acidosis risk and can rank barley samples based on their acidotic risk. Nonetheless, the model would benefit from further refinement by expanding the database.

Enumerating Hematopoietic Stem and Progenitor Cells in Zebrafish Embryos.

PubMed

Esain, Virginie; Cortes, Mauricio; North, Trista E

2016-01-01

Over the past 20 years, zebrafish have proven to be a valuable model to dissect the signaling pathways involved in hematopoiesis, including Hematopoietic Stem and Progenitor Cell (HSPC) formation and homeostasis. Despite tremendous efforts to generate the tools necessary to characterize HSPCs in vitro and in vivo the zebrafish community still lacks standardized methods to quantify HSPCs across laboratories. Here, we describe three methods used routinely in our lab, and in others, to reliably enumerate HSPCs in zebrafish embryos: large-scale live imaging of transgenic reporter lines, Fluorescence-Activated Cell Sorting (FACS), and in vitro cell culture. While live imaging and FACS analysis allows enumeration of total or site-specific HSPCs, the cell culture assay provides the unique opportunity to test the functional potential of isolated HSPCs, similar to those employed in mammals.

A method for evaluating nanoparticle transport through the blood-brain barrier in vitro.

PubMed

Guarnieri, Daniela; Muscetti, Ornella; Netti, Paolo A

2014-01-01

Blood-brain barrier (BBB) represents a formidable barrier for many therapeutic drugs to enter the brain tissue. The development of new strategies for enhancing drug delivery to the brain is of great importance in diagnostics and therapeutics of central nervous system (CNS) diseases. In this context, nanoparticles are an emerging class of drug delivery systems that can be easily tailored to deliver drugs to various compartments of the body, including the brain. To identify, characterize, and validate novel nanoparticles applicable to brain delivery, in vitro BBB model systems have been developed. In this work, we describe a method to screen nanoparticles with variable size and surface functionalization in order to define the physicochemical characteristics underlying the design of nanoparticles that are able to efficiently cross the BBB.

In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the Right Platform.

PubMed

Katt, Moriah E; Placone, Amanda L; Wong, Andrew D; Xu, Zinnia S; Searson, Peter C

2016-01-01

In vitro tumor models have provided important tools for cancer research and serve as low-cost screening platforms for drug therapies; however, cancer recurrence remains largely unchecked due to metastasis, which is the cause of the majority of cancer-related deaths. The need for an improved understanding of the progression and treatment of cancer has pushed for increased accuracy and physiological relevance of in vitro tumor models. As a result, in vitro tumor models have concurrently increased in complexity and their output parameters further diversified, since these models have progressed beyond simple proliferation, invasion, and cytotoxicity screens and have begun recapitulating critical steps in the metastatic cascade, such as intravasation, extravasation, angiogenesis, matrix remodeling, and tumor cell dormancy. Advances in tumor cell biology, 3D cell culture, tissue engineering, biomaterials, microfabrication, and microfluidics have enabled rapid development of new in vitro tumor models that often incorporate multiple cell types, extracellular matrix materials, and spatial and temporal introduction of soluble factors. Other innovations include the incorporation of perfusable microvessels to simulate the tumor vasculature and model intravasation and extravasation. The drive toward precision medicine has increased interest in adapting in vitro tumor models for patient-specific therapies, clinical management, and assessment of metastatic potential. Here, we review the wide range of current in vitro tumor models and summarize their advantages, disadvantages, and suitability in modeling specific aspects of the metastatic cascade and drug treatment.

In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the Right Platform

PubMed Central

Katt, Moriah E.; Placone, Amanda L.; Wong, Andrew D.; Xu, Zinnia S.; Searson, Peter C.

2016-01-01

In vitro tumor models have provided important tools for cancer research and serve as low-cost screening platforms for drug therapies; however, cancer recurrence remains largely unchecked due to metastasis, which is the cause of the majority of cancer-related deaths. The need for an improved understanding of the progression and treatment of cancer has pushed for increased accuracy and physiological relevance of in vitro tumor models. As a result, in vitro tumor models have concurrently increased in complexity and their output parameters further diversified, since these models have progressed beyond simple proliferation, invasion, and cytotoxicity screens and have begun recapitulating critical steps in the metastatic cascade, such as intravasation, extravasation, angiogenesis, matrix remodeling, and tumor cell dormancy. Advances in tumor cell biology, 3D cell culture, tissue engineering, biomaterials, microfabrication, and microfluidics have enabled rapid development of new in vitro tumor models that often incorporate multiple cell types, extracellular matrix materials, and spatial and temporal introduction of soluble factors. Other innovations include the incorporation of perfusable microvessels to simulate the tumor vasculature and model intravasation and extravasation. The drive toward precision medicine has increased interest in adapting in vitro tumor models for patient-specific therapies, clinical management, and assessment of metastatic potential. Here, we review the wide range of current in vitro tumor models and summarize their advantages, disadvantages, and suitability in modeling specific aspects of the metastatic cascade and drug treatment. PMID:26904541

In vitro and in vivo correlation of disintegration and bitter taste masking using orally disintegrating tablet containing ion exchange resin-drug complex.

PubMed

Kim, Jong-Il; Cho, Sang-Min; Cui, Jing-Hao; Cao, Qing-Ri; Oh, Euichaul; Lee, Beom-Jin

2013-10-15

Although the taste-masking of bitter drug using ion exchange resin has been recognized, in vitro testing using an electronic tongue (e-Tongue) and in vivo bitterness test by human panel test was not fully understood. In case of orally disintegrating tablet (ODT) containing bitter medicine, in vitro and in vivo disintegration is also importance for dosage performance. Donepezil hydrochloride was chosen as a model drug due to its bitterness and requires rapid disintegration for the preparation of ODT. In this study, ion exchange resin drug complex (IRDC) at three different ratios (1:2, 1:1, 2:1) was prepared using a spray-drying method and then IRDC-loaded ODT containing superdisintegrants (crospovidone, croscarmellose sodium, and sodium starch glycolate) were prepared by the direct compression method. The physical properties and morphologies were then characterized by scanning electron microscopy (SEM), X-ray powder diffraction (PXRD) and electrophoretic laser scattering (ELS), respectively. The in vitro taste-masking efficiency was measured with an electronic tongue (e-Tongue). In vivo bitterness scale was also evaluated by human volunteers and then we defined new term, "bitterness index (BI)" to link in vitro e-Tongue. There was a good correlation of IRDC between in vitro e-Tongue values and in vivo BI. Furthermore, IRDC-loaded ODT showed good in vitro/in vivo correlation in the disintegration time. The optimal IRDC-loaded ODTs displayed similar drug release profiles to the reference tablet (Aricept(®) ODT) in release media of pH 1.2, pH 4.0, pH 6.8 and distilled water but had significantly better palatability in vivo taste-masking evaluation. The current IRDC-loaded ODT according to the in vitro and in vivo correlation of disintegration and bitter taste masking could provide platforms in ODT dosage formulations of donepezil hydrochloride for improved patient compliances. Copyright © 2013 Elsevier B.V. All rights reserved.

Solid lipid nanoparticles by coacervation loaded with a methotrexate prodrug: preliminary study for glioma treatment.

PubMed

Battaglia, Luigi; Muntoni, Elisabetta; Chirio, Daniela; Peira, Elena; Annovazzi, Laura; Schiffer, Davide; Mellai, Marta; Riganti, Chiara; Salaroglio, Iris Chiara; Lanotte, Michele; Panciani, Pierpaolo; Capucchio, Maria Teresa; Valazza, Alberto; Biasibetti, Elena; Gallarate, Marina

2017-03-01

Methotrexate-loaded biocompatible nanoparticles were tested for preliminary efficacy in glioma treatment. Behenic acid nanoparticles, prepared by the coacervation method, were loaded with the ester prodrug didodecylmethotrexate, which was previously tested in vitro against glioblastoma human primary cultures. Nanoparticle conjugation with an ApoE mimicking chimera peptide was performed to obtain active targeting to the brain. Biodistribution studies in healthy rats assessed the superiority of ApoE-conjugated formulation, which was tested on an F98/Fischer glioma model. Differences were observed in tumor growth rate (measured by MRI) between control and treated rats. In vitro tests on F98 cultured cells assessed their susceptibility to treatment, with consequent apoptosis, and allowed us to explain the apoptosis observed in glioma models.

Range-Finding Risk Assessment of Inhalation Exposure to Nanodiamonds in a Laboratory Environment

PubMed Central

Koivisto, Antti J.; Palomäki, Jaana E.; Viitanen, Anna-Kaisa; Siivola, Kirsi M.; Koponen, Ismo K.; Yu, Mingzhou; Kanerva, Tomi S.; Norppa, Hannu; Alenius, Harri T.; Hussein, Tareq; Savolainen, Kai M.; Hämeri, Kaarle J.

2014-01-01

This study considers fundamental methods in occupational risk assessment of exposure to airborne engineered nanomaterials. We discuss characterization of particle emissions, exposure assessment, hazard assessment with in vitro studies, and risk range characterization using calculated inhaled doses and dose-response translated to humans from in vitro studies. Here, the methods were utilized to assess workers’ risk range of inhalation exposure to nanodiamonds (NDs) during handling and sieving of ND powder. NDs were agglomerated to over 500 nm particles, and mean exposure levels of different work tasks varied from 0.24 to 4.96 µg·m−3 (0.08 to 0.74 cm−3). In vitro-experiments suggested that ND exposure may cause a risk for activation of inflammatory cascade. However, risk range characterization based on in vitro dose-response was not performed because accurate assessment of delivered (settled) dose on the cells was not possible. Comparison of ND exposure with common pollutants revealed that ND exposure was below 5 μg·m−3, which is one of the proposed exposure limits for diesel particulate matter, and the workers’ calculated dose of NDs during the measurement day was 74 ng which corresponded to 0.02% of the modeled daily (24 h) dose of submicrometer urban air particles. PMID:24840353

Genetic toxicity assessment of engineered nanoparticles using a 3D in vitro skin model (EpiDerm™).

PubMed

Wills, John W; Hondow, Nicole; Thomas, Adam D; Chapman, Katherine E; Fish, David; Maffeis, Thierry G; Penny, Mark W; Brown, Richard A; Jenkins, Gareth J S; Brown, Andy P; White, Paul A; Doak, Shareen H

2016-09-09

The rapid production and incorporation of engineered nanomaterials into consumer products alongside research suggesting nanomaterials can cause cell death and DNA damage (genotoxicity) makes in vitro assays desirable for nanosafety screening. However, conflicting outcomes are often observed when in vitro and in vivo study results are compared, suggesting more physiologically representative in vitro models are required to minimise reliance on animal testing. BASF Levasil® silica nanoparticles (16 and 85 nm) were used to adapt the 3D reconstructed skin micronucleus (RSMN) assay for nanomaterials administered topically or into the growth medium. 3D dose-responses were compared to a 2D micronucleus assay using monocultured human B cells (TK6) after standardising dose between 2D / 3D assays by total nanoparticle mass to cell number. Cryogenic vitrification, scanning electron microscopy and dynamic light scattering techniques were applied to characterise in-medium and air-liquid interface exposures. Advanced transmission electron microscopy imaging modes (high angle annular dark field) and X-ray spectrometry were used to define nanoparticle penetration / cellular uptake in the intact 3D models and 2D monocultured cells. For all 2D exposures, significant (p < 0.002) increases in genotoxicity were observed (≥100 μg/mL) alongside cell viability decreases (p < 0.015) at doses ≥200 μg/mL (16 nm-SiO2) and ≥100 μg/mL (85 nm-SiO2). In contrast, 2D-equivalent exposures to the 3D models (≤300 μg/mL) caused no significant DNA damage or impact on cell viability. Further increasing dose to the 3D models led to probable air-liquid interface suffocation. Nanoparticle penetration / cell uptake analysis revealed no exposure to the live cells of the 3D model occurred due to the protective nature of the skin model's 3D cellular microarchitecture (topical exposures) and confounding barrier effects of the collagen cell attachment layer (in-medium exposures). 2D monocultured cells meanwhile showed extensive internalisation of both silica particles causing (geno)toxicity. The results establish the importance of tissue microarchitecture in defining nanomaterial exposure, and suggest 3D in vitro models could play a role in bridging the gap between in vitro and in vivo outcomes in nanotoxicology. Robust exposure characterisation and uptake assessment methods (as demonstrated) are essential to interpret nano(geno)toxicity studies successfully.

Chronic Modes of Action for Ecotoxicity: Integrated Testing Strategies for the 21st Century, presentation

EPA Science Inventory

This initiative, though focusing perhaps on individual assays at the beginning, recognizes the need for a tiered strategy for ecotoxicity tests that better integrates new techniques (models, in vitro methods, new technologies) to improve ecotoxicity testing as a whole. The reali...

NCCT ToxCast Program for Nanomaterial Prioritization: High-Throughput Screening, Consideration of Exposure, and Bioactivity Profiling/Modeling

EPA Science Inventory

Find relationships between bioactivities and NM characteristics or testing conditions. Recommend a dose metric for NMs in vitro studies. Establish associations to in vivo toxicity or pathways identified from testing of conventional chemicals with ToxCast HTS methods. May be abl...

Role of Metabolomics in Environmental Chemical Exposure and Risk Assessment

EPA Science Inventory

The increasing demand for the reduction, replacement, and refinement of the use of animal models in exposure assessments has stimulated the pursuit of alternative methods. This has included not only the use of the in vitro systems (e.g., cell cultures) in lieu of in vivo whole an...

Reduced expression IRF7 in nasal epithelial cells from smokers as a potential mechanism mediating enhanced susceptibility to influenza

EPA Science Inventory

Rationale: Smokers are more susceptible to viral infections, including influenza virus, yet the mechanisms mediating this effect are not known. Methods: We have established an in vitro model of differentiated nasal epithelial cells from smokers, which maintain enhanced levels...

EDC testing in the future: Exploring roles of pathway-based in silico, in vitro and in vivo methods

EPA Science Inventory

Many thoroughly validated, robust tests with both mammalian and non-mammalian models have been developed to identify chemicals with the potential to impact endocrine pathways associated with the hypothalamic-pituitary-gonadal (HPG) and thyroidal axes. In the US, for example, the...

Toxicokinetic Triage for Environmental Chemicals

PubMed Central

Wambaugh, John F.; Wetmore, Barbara A.; Pearce, Robert; Strope, Cory; Goldsmith, Rocky; Sluka, James P.; Sedykh, Alexander; Tropsha, Alex; Bosgra, Sieto; Shah, Imran; Judson, Richard; Thomas, Russell S.; Woodrow Setzer, R.

2015-01-01

Toxicokinetic (TK) models link administered doses to plasma, blood, and tissue concentrations. High-throughput TK (HTTK) performs in vitro to in vivo extrapolation to predict TK from rapid in vitro measurements and chemical structure-based properties. A significant toxicological application of HTTK has been “reverse dosimetry,” in which bioactive concentrations from in vitro screening studies are converted into in vivo doses (mg/kg BW/day). These doses are predicted to produce steady-state plasma concentrations that are equivalent to in vitro bioactive concentrations. In this study, we evaluate the impact of the approximations and assumptions necessary for reverse dosimetry and develop methods to determine whether HTTK tools are appropriate or may lead to false conclusions for a particular chemical. Based on literature in vivo data for 87 chemicals, we identified specific properties (eg, in vitro HTTK data, physico-chemical descriptors, and predicted transporter affinities) that correlate with poor HTTK predictive ability. For 271 chemicals we developed a generic HT physiologically based TK (HTPBTK) model that predicts non-steady-state chemical concentration time-courses for a variety of exposure scenarios. We used this HTPBTK model to find that assumptions previously used for reverse dosimetry are usually appropriate, except most notably for highly bioaccumulative compounds. For the thousands of man-made chemicals in the environment that currently have no TK data, we propose a 4-element framework for chemical TK triage that can group chemicals into 7 different categories associated with varying levels of confidence in HTTK predictions. For 349 chemicals with literature HTTK data, we differentiated those chemicals for which HTTK approaches are likely to be sufficient, from those that may require additional data. PMID:26085347

An untargeted multi-technique metabolomics approach to studying intracellular metabolites of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

PubMed

Ruiz-Aracama, Ainhoa; Peijnenburg, Ad; Kleinjans, Jos; Jennen, Danyel; van Delft, Joost; Hellfrisch, Caroline; Lommen, Arjen

2011-05-20

In vitro cell systems together with omics methods represent promising alternatives to conventional animal models for toxicity testing. Transcriptomic and proteomic approaches have been widely applied in vitro but relatively few studies have used metabolomics. Therefore, the goal of the present study was to develop an untargeted methodology for performing reproducible metabolomics on in vitro systems. The human liver cell line HepG2, and the well-known hepatotoxic and non-genotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were used as the in vitro model system and model toxicant, respectively. The study focused on the analysis of intracellular metabolites using NMR, LC-MS and GC-MS, with emphasis on the reproducibility and repeatability of the data. State of the art pre-processing and alignment tools and multivariate statistics were used to detect significantly altered levels of metabolites after exposing HepG2 cells to TCDD. Several metabolites identified using databases, literature and LC-nanomate-Orbitrap analysis were affected by the treatment. The observed changes in metabolite levels are discussed in relation to the reported effects of TCDD. Untargeted profiling of the polar and apolar metabolites of in vitro cultured HepG2 cells is a valid approach to studying the effects of TCDD on the cell metabolome. The approach described in this research demonstrates that highly reproducible experiments and correct normalization of the datasets are essential for obtaining reliable results. The effects of TCDD on HepG2 cells reported herein are in agreement with previous studies and serve to validate the procedures used in the present work.

Biohybrid Membrane Systems and Bioreactors as Tools for In Vitro Drug Testing.

PubMed

Salerno, Simona; Bartolo, Loredana De

2017-01-01

In drug development, in vitro human model systems are absolutely essential prior to the clinical trials, considering the increasing number of chemical compounds in need of testing, and, keeping in mind that animals cannot predict all the adverse human health effects and reactions, due to the species-specific differences in metabolic pathways. The liver plays a central role in the clearance and biotransformation of chemicals and xenobiotics. In vitro liver model systems by using highly differentiated human cells could have a great impact in preclinical trials. Membrane biohybrid systems constituted of human hepatocytes and micro- and nano-structured membranes, represent valuable tools for studying drug metabolism and toxicity. Membranes act as an extracellular matrix for the adhesion of hepatocytes, and compartmentalise them in a well-defined physical and chemical microenvironment with high selectivity. Advanced 3-D tissue cultures are furthermore achieved by using membrane bioreactors (MBR), which ensure the continuous perfusion of cells protecting them from shear stress. MBRs with different configurations allow the culturing of cells at high density and under closely monitored high perfusion, similarly to the natural liver. These devices that promote the long-term maintenance and differentiation of primary human hepatocytes with preserved liver specific functions can be employed in drug testing for prolonged exposure to chemical compounds and for assessing repeated-dose toxicity. The use of primary human hepatocytes in MBRs is the only system providing a faster and more cost-effective method of analysis for the prediction of in vitro human drug metabolism and enzyme induction alternative and/or complementary to the animal experimentation. In this paper, in vitro models for studying drug metabolism and toxicity as advanced biohybrid membrane systems and MBRs will be reviewed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

vFitness: a web-based computing tool for improving estimation of in vitro HIV-1 fitness experiments

PubMed Central

2010-01-01

Background The replication rate (or fitness) between viral variants has been investigated in vivo and in vitro for human immunodeficiency virus (HIV). HIV fitness plays an important role in the development and persistence of drug resistance. The accurate estimation of viral fitness relies on complicated computations based on statistical methods. This calls for tools that are easy to access and intuitive to use for various experiments of viral fitness. Results Based on a mathematical model and several statistical methods (least-squares approach and measurement error models), a Web-based computing tool has been developed for improving estimation of virus fitness in growth competition assays of human immunodeficiency virus type 1 (HIV-1). Conclusions Unlike the two-point calculation used in previous studies, the estimation here uses linear regression methods with all observed data in the competition experiment to more accurately estimate relative viral fitness parameters. The dilution factor is introduced for making the computational tool more flexible to accommodate various experimental conditions. This Web-based tool is implemented in C# language with Microsoft ASP.NET, and is publicly available on the Web at http://bis.urmc.rochester.edu/vFitness/. PMID:20482791

vFitness: a web-based computing tool for improving estimation of in vitro HIV-1 fitness experiments.

PubMed

Ma, Jingming; Dykes, Carrie; Wu, Tao; Huang, Yangxin; Demeter, Lisa; Wu, Hulin

2010-05-18

The replication rate (or fitness) between viral variants has been investigated in vivo and in vitro for human immunodeficiency virus (HIV). HIV fitness plays an important role in the development and persistence of drug resistance. The accurate estimation of viral fitness relies on complicated computations based on statistical methods. This calls for tools that are easy to access and intuitive to use for various experiments of viral fitness. Based on a mathematical model and several statistical methods (least-squares approach and measurement error models), a Web-based computing tool has been developed for improving estimation of virus fitness in growth competition assays of human immunodeficiency virus type 1 (HIV-1). Unlike the two-point calculation used in previous studies, the estimation here uses linear regression methods with all observed data in the competition experiment to more accurately estimate relative viral fitness parameters. The dilution factor is introduced for making the computational tool more flexible to accommodate various experimental conditions. This Web-based tool is implemented in C# language with Microsoft ASP.NET, and is publicly available on the Web at http://bis.urmc.rochester.edu/vFitness/.

Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project.

PubMed

de Hoogt, Ronald; Estrada, Marta F; Vidic, Suzana; Davies, Emma J; Osswald, Annika; Barbier, Michael; Santo, Vítor E; Gjerde, Kjersti; van Zoggel, Hanneke J A A; Blom, Sami; Dong, Meng; Närhi, Katja; Boghaert, Erwin; Brito, Catarina; Chong, Yolanda; Sommergruber, Wolfgang; van der Kuip, Heiko; van Weerden, Wytske M; Verschuren, Emmy W; Hickman, John; Graeser, Ralph

2017-11-21

Two-dimensional (2D) culture of cancer cells in vitro does not recapitulate the three-dimensional (3D) architecture, heterogeneity and complexity of human tumors. More representative models are required that better reflect key aspects of tumor biology. These are essential studies of cancer biology and immunology as well as for target validation and drug discovery. The Innovative Medicines Initiative (IMI) consortium PREDECT (www.predect.eu) characterized in vitro models of three solid tumor types with the goal to capture elements of tumor complexity and heterogeneity. 2D culture and 3D mono- and stromal co-cultures of increasing complexity, and precision-cut tumor slice models were established. Robust protocols for the generation of these platforms are described. Tissue microarrays were prepared from all the models, permitting immunohistochemical analysis of individual cells, capturing heterogeneity. 3D cultures were also characterized using image analysis. Detailed step-by-step protocols, exemplary datasets from the 2D, 3D, and slice models, and refined analytical methods were established and are presented.

Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project

PubMed Central

de Hoogt, Ronald; Estrada, Marta F.; Vidic, Suzana; Davies, Emma J.; Osswald, Annika; Barbier, Michael; Santo, Vítor E.; Gjerde, Kjersti; van Zoggel, Hanneke J. A. A.; Blom, Sami; Dong, Meng; Närhi, Katja; Boghaert, Erwin; Brito, Catarina; Chong, Yolanda; Sommergruber, Wolfgang; van der Kuip, Heiko; van Weerden, Wytske M.; Verschuren, Emmy W.; Hickman, John; Graeser, Ralph

2017-01-01

Two-dimensional (2D) culture of cancer cells in vitro does not recapitulate the three-dimensional (3D) architecture, heterogeneity and complexity of human tumors. More representative models are required that better reflect key aspects of tumor biology. These are essential studies of cancer biology and immunology as well as for target validation and drug discovery. The Innovative Medicines Initiative (IMI) consortium PREDECT (www.predect.eu) characterized in vitro models of three solid tumor types with the goal to capture elements of tumor complexity and heterogeneity. 2D culture and 3D mono- and stromal co-cultures of increasing complexity, and precision-cut tumor slice models were established. Robust protocols for the generation of these platforms are described. Tissue microarrays were prepared from all the models, permitting immunohistochemical analysis of individual cells, capturing heterogeneity. 3D cultures were also characterized using image analysis. Detailed step-by-step protocols, exemplary datasets from the 2D, 3D, and slice models, and refined analytical methods were established and are presented. PMID:29160867

Can in vitro assays account for interactions between inorganic co-contaminants observed during in vivo relative bioavailability assessment?

PubMed

Ollson, Cameron J; Smith, Euan; Juhasz, Albert L

2018-02-01

In vitro assays act as surrogate measurements of relative bioavailability (RBA) for inorganic contaminants. The values derived from these assays are routinely used to refine human health risk assessments (HHRA). Extensive in vitro research has been performed on three major inorganic contaminants; As, Cd and Pb. However, the majority of these studies have evaluated the contaminants individually, even in cases when they are found as co-contaminants. Recently, in vivo studies (animal model) have determined that when the three aforementioned contaminants are present in the same soil matrix, they have the ability to influence each other's individual bioavailability. Since in vitro assays are used to inform HHRA, this study investigated whether bioaccessibility methods including the Solubility/Bioavailability Research Consortium (SBRC) assay, and physiologically based extraction test (PBET), have the ability to detect interactions between As, Cd and Pb. Using a similar dosing methodology to recently published in vivo studies, spiked aged (12 years) soil was assessed by evaluating contaminant bioaccessibility individually, in addition to tertiary combinations. In two spiked aged soils (grey and brown chromosols), there was no influence on contaminant bioaccessibility when As, Cd and Pb we present as co-contaminants. However, in a red ferrosol, the presence of As and Pb significantly decreased (p < 0.05) the bioaccessibility of Cd when assessed using gastric and intestinal phases of the SBRC assay and the PBET. Conceivable, differences in key physico-chemical properties (TOC, Fe, Al, P) between the study soils influenced contaminant interactions and bioaccessibility outcomes. Although bioaccessibility methods may not account for interactions between elements as demonstrated in in vivo models, in vitro assessment provides a conservative prediction of contaminant RBA under co-contaminant scenarios. Copyright © 2017 Elsevier Ltd. All rights reserved.

[In silico, in vitro, in omic experimental models and drug safety evaluation].

PubMed

Claude, Nancy; Goldfain-Blanc, Françoise; Guillouzo, André

2009-01-01

Over the last few decades, toxicology has benefited from scientific, technical, and bioinformatic developments relating to patient safety assessment during clinical and drug marketing studies. Based on this knowledge, new in silico, in vitro, and "omic" experimental models are emerging. Although these models cannot currently replace classic safety evaluations performed on laboratory animals, they allow compounds with unacceptable toxicity to be rejected in the early stages of drug development, thereby reducing the number of laboratory animals needed. In addition, because these models are particularly adapted to mechanistic studies, they can help to improve the relevance of the data obtained, thus enabling better prevention and screening of the adverse effects that may occur in humans. Much progress remains to be done, especially in the field of validation. Nevertheless, current efforts by industrial, academic laboratories, and regulatory agencies should, in coming years, significantly improve preclinical drug safety evaluation thanks to the integration of these new methods into the drug research and development process.

Condensational Growth of Combination Drug-Excipient Submicrometer Particles for Targeted High Efficiency Pulmonary Delivery: Comparison of CFD Predictions with Experimental Results

PubMed Central

Hindle, Michael

2011-01-01

Purpose The objective of this study was to investigate the hygroscopic growth of combination drug and excipient submicrometer aerosols for respiratory drug delivery using in vitro experiments and a newly developed computational fluid dynamics (CFD) model. Methods Submicrometer combination drug and excipient particles were generated experimentally using both the capillary aerosol generator and the Respimat inhaler. Aerosol hygroscopic growth was evaluated in vitro and with CFD in a coiled tube geometry designed to provide residence times and thermodynamic conditions consistent with the airways. Results The in vitro results and CFD predictions both indicated that the initially submicrometer particles increased in mean size to a range of 1.6–2.5 µm for the 50:50 combination of a non-hygroscopic drug (budesonide) and different hygroscopic excipients. CFD results matched the in vitro predictions to within 10% and highlighted gradual and steady size increase of the droplets, which will be effective for minimizing extrathoracic deposition and producing deposition deep within the respiratory tract. Conclusions Enhanced excipient growth (EEG) appears to provide an effective technique to increase pharmaceutical aerosol size, and the developed CFD model will provide a powerful design tool for optimizing this technique to produce high efficiency pulmonary delivery. PMID:21948458

Evaluation of in-vitro antibacterial activity and anti-inflammatory activity for different extracts of Rauvolfia tetraphylla L. root bark

PubMed Central

Ganga Rao, B.; Umamaheswara Rao, P.; Sambasiva Rao, E.; Mallikarjuna Rao, T.; Praneeth. D, V. S.

2012-01-01

Objective To assess the in-vitro antibacterial activity and anti-inflammatory activity of orally administered different extracts (Hydro-alcoholic, methanolic, ethyl acetate and hexane) of Rauvolfia tetraphylla (R. tetraphylla) root bark in Carrageenan induced acute inflammation in rats. Methods In-vitro antibacterial activity was evaluated for extracts against four Gram positive and four Gram negative bacteria by using cylinder plate assay. Hydro-alcoholic extract (70% v/v ethanol) at 200, 400 and 800 mg/kg doses and methanolic, ethyl acetate and hexane extracts at doses 100, 200 and 400 mg/kg were tested for anti-inflammatory activity in Carrageenan induced rat paw oedema model and paw thickness was measured every one hour up to 6 hrs. Results All extracts of R. tetraphylla root bark showed good zone of inhibition against tested bacterial strains. In Carrageenan induced inflammation model, hydro-alcoholic and methanolic extract of R. tetraphylla root bark at three different doses produced significant (P<0.001) reduction when compared to vehicle treated control group and hexane, ethyl acetate extracts. Conclusions In the present study extracts of R. tetraphylla root bark shows good in-vitro antibacterial activity and in-vivo anti-inflammatory activity in rats. PMID:23569853

ToxCast Phase I

EPA Pesticide Factsheets

Background: Chemical toxicity testing is being transformed by advances in biology and computer modeling, concerns over animal use and the thousands of environmental chemicals lacking toxicity data. EPA's ToxCast program aims to address these concerns by screening and prioritizing chemicals for potential human toxicity using in vitro assays and in silico approaches. Objectives: This project aims to evaluate the use of in vitro assays for understanding the types of molecular and pathway perturbations caused by environmental chemicals and to build initial prioritization models of in vivo toxicity. Methods: We tested 309 mostly pesticide active chemicals in 467 assays across 9 technologies, including high-throughput cell-free assays and cell-based assays in multiple human primary cells and cell lines, plus rat primary hepatocytes. Both individual and composite scores for effects on genes and pathways were analyzed. Results: Chemicals display a broad spectrum of activity at the molecular and pathway levels. Many expected interactions are seen, including endocrine and xenobiotic metabolism enzyme activity. Chemicals range in promiscuity across pathways, from no activity to affecting dozens of pathways. We find a statistically significant inverse association between the number of pathways perturbed by a chemical at low in vitro concentrations and the lowest in vivo dose at which a chemical causes toxicity. We also find associations between a small set in vitro ass

Tissue vascularization through 3D printing: Will technology bring us flow?

PubMed

Paulsen, S J; Miller, J S

2015-05-01

Though in vivo models provide the most physiologically relevant environment for studying tissue function, in vitro studies provide researchers with explicit control over experimental conditions and the potential to develop high throughput testing methods. In recent years, advancements in developmental biology research and imaging techniques have significantly improved our understanding of the processes involved in vascular development. However, the task of recreating the complex, multi-scale vasculature seen in in vivo systems remains elusive. 3D bioprinting offers a potential method to generate controlled vascular networks with hierarchical structure approaching that of in vivo networks. Bioprinting is an interdisciplinary field that relies on advances in 3D printing technology along with advances in imaging and computational modeling, which allow researchers to monitor cellular function and to better understand cellular environment within the printed tissue. As bioprinting technologies improve with regards to resolution, printing speed, available materials, and automation, 3D printing could be used to generate highly controlled vascularized tissues in a high throughput manner for use in regenerative medicine and the development of in vitro tissue models for research in developmental biology and vascular diseases. © 2015 Wiley Periodicals, Inc.

In silico and in vitro methods to optimize the performance of experimental gastroretentive floating mini-tablets.

PubMed

Eberle, Veronika A; Häring, Armella; Schoelkopf, Joachim; Gane, Patrick A C; Huwyler, Jörg; Puchkov, Maxim

2016-01-01

Development of floating drug delivery systems (FDDS) is challenging. To facilitate this task, an evaluation method was proposed, which allows for a combined investigation of drug release and flotation. It was the aim of the study to use functionalized calcium carbonate (FCC)-based lipophilic mini-tablet formulations as a model system to design FDDS with a floating behavior characterized by no floating lag time, prolonged flotation and loss of floating capability after complete drug release. Release of the model drug caffeine from the mini-tablets was assessed in vitro by a custom-built stomach model. A cellular automata-based model was used to simulate tablet dissolution. Based on the in silico data, floating forces were calculated and analyzed as a function of caffeine release. Two floating behaviors were identified for mini-tablets: linear decrease of the floating force and maintaining of the floating capability until complete caffeine release. An optimal mini-tablet formulation with desired drug release time and floating behavior was developed and tested. A classification system for a range of varied floating behavior of FDDS was proposed. The FCC-based mini-tablets had an ideal floating behavior: duration of flotation is defined and floating capability decreases after completion of drug release.

A Novel Two-Step Hierarchial Quantitative Structure-Activity ...

EPA Pesticide Factsheets

Background: Accurate prediction of in vivo toxicity from in vitro testing is a challenging problem. Large public–private consortia have been formed with the goal of improving chemical safety assessment by the means of high-throughput screening. Methods and results: A database containing experimental cytotoxicity values for in vitro half-maximal inhibitory concentration (IC50) and in vivo rodent median lethal dose (LD50) for more than 300 chemicals was compiled by Zentralstelle zur Erfassung und Bewertung von Ersatz- und Ergaenzungsmethoden zum Tierversuch (ZEBET ; National Center for Documentation and Evaluation of Alternative Methods to Animal Experiments) . The application of conventional quantitative structure–activity relationship (QSAR) modeling approaches to predict mouse or rat acute LD50 values from chemical descriptors of ZEBET compounds yielded no statistically significant models. The analysis of these data showed no significant correlation between IC50 and LD50. However, a linear IC50 versus LD50 correlation could be established for a fraction of compounds. To capitalize on this observation, we developed a novel two-step modeling approach as follows. First, all chemicals are partitioned into two groups based on the relationship between IC50 and LD50 values: One group comprises compounds with linear IC50 versus LD50 relationships, and another group comprises the remaining compounds. Second, we built conventional binary classification QSAR models t

Polymeric nanoparticles containing diazepam: preparation, optimization, characterization, in-vitro drug release and release kinetic study

NASA Astrophysics Data System (ADS)

Bohrey, Sarvesh; Chourasiya, Vibha; Pandey, Archna

2016-03-01

Nanoparticles formulated from biodegradable polymers like poly(lactic-co-glycolic acid) (PLGA) are being extensively investigated as drug delivery systems due to their two important properties such as biocompatibility and controlled drug release characteristics. The aim of this work to formulated diazepam loaded PLGA nanoparticles by using emulsion solvent evaporation technique. Polyvinyl alcohol (PVA) is used as stabilizing agent. Diazepam is a benzodiazepine derivative drug, and widely used as an anticonvulsant in the treatment of various types of epilepsy, insomnia and anxiety. This work investigates the effects of some preparation variables on the size and shape of nanoparticles prepared by emulsion solvent evaporation method. These nanoparticles were characterized by photon correlation spectroscopy (PCS), transmission electron microscopy (TEM). Zeta potential study was also performed to understand the surface charge of nanoparticles. The drug release from drug loaded nanoparticles was studied by dialysis bag method and the in vitro drug release data was also studied by various kinetic models. The results show that sonication time, polymer content, surfactant concentration, ratio of organic to aqueous phase volume, and the amount of drug have an important effect on the size of nanoparticles. Hopefully we produced spherical shape Diazepam loaded PLGA nanoparticles with a size range under 250 nm with zeta potential -23.3 mV. The in vitro drug release analysis shows sustained release of drug from nanoparticles and follow Korsmeyer-Peppas model.

In vitro methods of assessing ocular biocompatibility using THP-1-derived macrophages.

PubMed

McCanna, David Joseph; Barthod-Malat, Aurore V; Gorbet, Maud B

2015-01-01

Macrophages play an important role in the elimination of infections, the removal of debris and in tissue repair after infection and trauma. In vitro models that assess ocular biomaterials for toxicity typically focus on the effects of these materials on epithelial or fibroblast cells. This investigation evaluated known ocular toxins deposited on model materials for their effects on the viability and activation of macrophages. THP-1-derived macrophages were cultured onto silicone films (used as a base biomaterial) deposited with chemical toxins (benzalkonium chloride (BAK), zinc diethyldithiocarbamate (ZDEC) and lipopolysaccharide (LPS)). Utilizing three fluorescent dyes calcein, ethidium homodimer-1 (EthD-1) and annexin V, the viability of macrophages attached to the biomaterial was determined using confocal microscopy. Propidium iodide (PI) staining and alamarBlue® (resazurin) reduction were used to assess cell death and metabolic activity. CD14, CD16, CD33, CD45, and CD54 expression of adherent macrophages, were also evaluated to detect LPS activation of macrophages using flow cytometry. The sensitivity of this test battery was demonstrated as significant toxicity from treated surfaces with ZDEC (0.001-0.01%), and BAK (0.001%-0.1%) was detected. Also, macrophage activation could be detected by measuring CD54 expression after exposure to adsorbed LPS. These in vitro methods will be helpful in determining the toxicity potential of new ocular biomaterials.

An ovarian bioreactor for in vitro culture of the whole bovine ovary: a preliminary report.

PubMed

Zanotelli, Matthew R; Henningsen, Joseph D; Hopkins, Patrick M; Dederich, Aaron P; Herman, Tessa; Puccinelli, Tracy J; Salih, Sana M

2016-08-04

Improved cancer therapeutics and enhanced cancer survivorship have emphasized the severe long-term side effects of chemotherapy. Specifically, studies have linked many chemotherapy agents with primary ovarian insufficiency, although an exact insult model has not yet been determined. To investigate and ultimately solve this problem, a novel device for extended study of mammalian ovaries in vitro was developed. A bioreactor was fabricated for bovine ovarian culture that provides intravascular delivery of media to the ovary through isolation and cannulation of a main ovarian artery branch. Whole ovaries were cultured in vitro using three methods: (1) continuously supplied fresh culture media, (2) recirculated culture media, or (3) continuously supplied fresh culture media supplemented with 500 nM doxorubicin for 24 or 48 h. TUNEL assay was used to assess apoptotic cell percentages in the three groups as compared to uncultured baseline ovaries. The ovary culture method was shown to maintain cell viability by effectively delivering nutrient-enriched pH-balanced media at a constant flow rate. Lower apoptosis observed in ovaries cultured in continuously supplied fresh culture media illustrates that this culture device and method are the first to sustain whole bovine ovary viability for 48 h. Meanwhile, the increase in the percentage of cell apoptosis with doxorubicin treatment indicates that the device can provide an alternative model for testing chemotherapy and chemoprotection treatments to prevent primary ovarian insufficiency in cancer patients. An ovarian bioreactor with consistent culture media flow through an ovarian vasculature-assisted approach maintains short-term whole bovine ovary viability.

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

PubMed Central

Dong, Yifei; Arif, Arif A.; Poon, Grace F. T.; Hardman, Blair; Dosanjh, Manisha; Johnson, Pauline

2016-01-01

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function. PMID:27404290

Integrated Model of Chemical Perturbations of a Biological Pathway Using 18 In Vitro High-Throughput Screening Assays for the Estrogen Receptor

PubMed Central

Judson, Richard S.; Magpantay, Felicia Maria; Chickarmane, Vijay; Haskell, Cymra; Tania, Nessy; Taylor, Jean; Xia, Menghang; Huang, Ruili; Rotroff, Daniel M.; Filer, Dayne L.; Houck, Keith A.; Martin, Matthew T.; Sipes, Nisha; Richard, Ann M.; Mansouri, Kamel; Setzer, R. Woodrow; Knudsen, Thomas B.; Crofton, Kevin M.; Thomas, Russell S.

2015-01-01

We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation, and ER-dependent cell proliferation. The network model uses activity patterns across the in vitro assays to predict whether a chemical is an ER agonist or antagonist, or is otherwise influencing the assays through a manner dependent on the physics and chemistry of the technology platform (“assay interference”). The method is applied to a library of 1812 commercial and environmental chemicals, including 45 ER positive and negative reference chemicals. Among the reference chemicals, the network model correctly identified the agonists and antagonists with the exception of very weak compounds whose activity was outside the concentration range tested. The model agonist score also correlated with the expected potency class of the active reference chemicals. Of the 1812 chemicals evaluated, 111 (6.1%) were predicted to be strongly ER active in agonist or antagonist mode. This dataset and model were also used to begin a systematic investigation of assay interference. The most prominent cause of false-positive activity (activity in an assay that is likely not due to interaction of the chemical with ER) is cytotoxicity. The model provides the ability to prioritize a large set of important environmental chemicals with human exposure potential for additional in vivo endocrine testing. Finally, this model is generalizable to any molecular pathway for which there are multiple upstream and downstream assays available. PMID:26272952

Toxicity hazard of organophosphate insecticide malathion identified by in vitro methods.

PubMed

Jira, David; Janousek, Stanislav; Pikula, Jiri; Vitula, Frantisek; Kejlova, Kristina

2012-01-01

Malathion is generally not classified as toxic. However, the toxicity seems to be species-dependent. Local and systemic toxicity data for birds are rare, but a decrease of wild bird densities in areas where malathion was applied was reported. Aim of the study was to extend knowledge on malathion toxicity on cellular and organ level and to evaluate embryotoxicity and genotoxicity for birds using the chick embryo model HET-CAM. Skin and eye irritation was determined using reconstructed skin and eye cornea tissues and the chorioallantoic membrane of chick embryo to simulate conjunctiva. Cytotoxicity in 3T3 Balb/c fibroblast culture was determined to estimate acute systemic toxicity. Chick embryo model was further employed to evaluate acute embryotoxicity for birds (mortality and genotoxicity). Data were analysed by means of general linear models. Malathion is not a skin and eye irritant. Cytotoxicity in vitro test provided LD50 value of 616 mg/kg suggesting higher toxic potential than is generally published based on in vivo tests on laboratory rodents. Embryotoxicity studies revealed dose and age dependent mortality of chick embryos. Genotoxicity was identified by means of micronucleus test in erythroid cells isolated from chorioallantois vascular system of chick embryos. Using in vitro alternative toxicological methods, a higher toxic potential of malathion was demonstrated than is generally declared. An increased health and environmental hazard may occur in areas with intensive agricultural production. The environmental consequences of delayed effects and embryotoxicity for bird populations in areas exposed to organophosphate insecticides, such as malathion, are obvious.

Characterization of primary human mammary epithelial cells isolated and propagated by conditional reprogrammed cell culture.

PubMed

Jin, Liting; Qu, Ying; Gomez, Liliana J; Chung, Stacey; Han, Bingchen; Gao, Bowen; Yue, Yong; Gong, Yiping; Liu, Xuefeng; Amersi, Farin; Dang, Catherine; Giuliano, Armando E; Cui, Xiaojiang

2018-02-20

Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro . We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.

New Alcohol and Onyx Mixture for Embolization: Feasibility and Proof of Concept in Both In Vitro and In Vivo Models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saeed Kilani, Mohammad, E-mail: msaeedkilani@gmail.com, E-mail: mohammadalikilani@yahoo.com; Zehtabi, Fatemeh, E-mail: fatemeh.zehtabi@gmail.com; Lerouge, Sophie, E-mail: Sophie.Lerouge@etsmtl.ca

IntroductionOnyx and ethanol are well-known embolic and sclerotic agents that are frequently used in embolization. These agents present advantages and disadvantages regarding visibility, injection control and penetration depth. Mixing both products might yield a new product with different characteristics. The aim of this study is to evaluate the injectability, radiopacity, and mechanical and occlusive properties of different mixtures of Onyx 18 and ethanol in vitro and in vivo (in a swine model).Materials and MethodsVarious Onyx 18 and ethanol formulations were prepared and tested in vitro for their injectability, solidification rate and shrinkage, cohesion and occlusive properties. In vivo tests weremore » performed using 3 swine. Ease of injection, radiopacity, cohesiveness and penetration were analyzed using fluoroscopy and high-resolution CT.ResultsAll mixtures were easy to inject through a microcatheter with no resistance or blockage in vitro and in vivo. The 50%-ethanol mixture showed delayed copolymerization with fragmentation and proximal occlusion. The 75%-ethanol mixture showed poor radiopacity in vivo and was not tested in vitro. The 25%-ethanol mixture showed good occlusive properties and accepted penetration and radiopacity.ConclusionMixing Onyx and ethanol is feasible. The mixture of 25% of ethanol and 75% of Onyx 18 could be a new sclero-embolic agent. Further research is needed to study the chemical changes of the mixture, to confirm the significance of the added sclerotic effect and to find out the ideal mixture percentages.« less

In Vitro Simulation and Validation of the Circulation with Congenital Heart Defects

PubMed Central

Figliola, Richard S.; Giardini, Alessandro; Conover, Tim; Camp, Tiffany A.; Biglino, Giovanni; Chiulli, John; Hsia, Tain-Yen

2010-01-01

Despite the recent advances in computational modeling, experimental simulation of the circulation with congenital heart defect using mock flow circuits remains an important tool for device testing, and for detailing the probable flow consequences resulting from surgical and interventional corrections. Validated mock circuits can be applied to qualify the results from novel computational models. New mathematical tools, coupled with advanced clinical imaging methods, allow for improved assessment of experimental circuit performance relative to human function, as well as the potential for patient-specific adaptation. In this review, we address the development of three in vitro mock circuits specific for studies of congenital heart defects. Performance of an in vitro right heart circulation circuit through a series of verification and validation exercises is described, including correlations with animal studies, and quantifying the effects of circuit inertiance on test results. We present our experience in the design of mock circuits suitable for investigations of the characteristics of the Fontan circulation. We use one such mock circuit to evaluate the accuracy of Doppler predictions in the presence of aortic coarctation. PMID:21218147

Comparative analysis of tumor spheroid generation techniques for differential in vitro drug toxicity

PubMed Central

Raghavan, Shreya; Rowley, Katelyn R.; Mehta, Geeta

2016-01-01

Multicellular tumor spheroids are powerful in vitro models to perform preclinical chemosensitivity assays. We compare different methodologies to generate tumor spheroids in terms of resultant spheroid morphology, cellular arrangement and chemosensitivity. We used two cancer cell lines (MCF7 and OVCAR8) to generate spheroids using i) hanging drop array plates; ii) liquid overlay on ultra-low attachment plates; iii) liquid overlay on ultra-low attachment plates with rotating mixing (nutator plates). Analysis of spheroid morphometry indicated that cellular compaction was increased in spheroids generated on nutator and hanging drop array plates. Collagen staining also indicated higher compaction and remodeling in tumor spheroids on nutator and hanging drop arrays compared to conventional liquid overlay. Consequently, spheroids generated on nutator or hanging drop plates had increased chemoresistance to cisplatin treatment (20-60% viability) compared to spheroids on ultra low attachment plates (10-20% viability). Lastly, we used a mathematical model to demonstrate minimal changes in oxygen and cisplatin diffusion within experimentally generated spheroids. Our results demonstrate that in vitro methods of tumor spheroid generation result in varied cellular arrangement and chemosensitivity. PMID:26918944

Assessment of in vitro COPD models for tobacco regulatory science: Workshop proceedings, conclusions and paths forward for in vitro model use.

PubMed

Behrsing, Holger; Raabe, Hans; Manuppello, Joseph; Bombick, Betsy; Curren, Rodger; Sullivan, Kristie; Sethi, Sanjay; Phipps, Richard; Tesfaigzi, Yohannes; Yan, Sherwin; D'Ruiz, Carl; Tarran, Robert; Constant, Samuel; Phillips, Gary; Gaça, Marianna; Hayden, Patrick; Cao, Xuefei; Mathis, Carole; Hoeng, Julia; Braun, Armin; Hill, Erin

2016-05-01

The Family Smoking Prevention and Tobacco Control Act of 2009 established the Food and Drug Administration Center for Tobacco Products (FDA-CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 8-10 December 2014, IIVS organised a workshop conference, entitled Assessment of In Vitro COPD Models for Tobacco Regulatory Science, to bring together stakeholders representing regulatory agencies, academia, industry and animal protection, to address the research priorities articulated by the FDA-CTP. Specific topics were covered to assess the status of current in vitro technologies as they are applied to understanding the adverse pulmonary events resulting from tobacco product exposure, and in particular, the progression of chronic obstructive pulmonary disease (COPD). The four topics covered were: a) Inflammation and Oxidative Stress; b) Ciliary Dysfunction and Ion Transport; c) Goblet Cell Hyperplasia and Mucus Production; and d) Parenchymal/Bronchial Tissue Destruction and Remodelling. The 2.5 day workshop included 18 expert speakers, plus poster sessions, networking and breakout sessions, which identified key findings and provided recommendations to advance the in vitro technologies and assays used to evaluate tobacco-induced disease etiologies. The workshop summary was reported at the 2015 Society of Toxicology Annual Meeting, and the recommendations led to an IIVS-organised technical workshop in June 2015, entitled Goblet Cell Hyperplasia, Mucus Production, and Ciliary Beating Assays, to assess these assays and to conduct a proof-of-principle multi-laboratory exercise to determine their suitability for standardisation. Here, we report on the proceedings, recommendations and outcomes of the December 2014 workshop, including paths forward to continue the development of non-animal methods to evaluate tissue responses that model the disease processes that may lead to COPD, a major cause of mortality worldwide. 2016 FRAME.

Development and validation of an automated and marker-free CT-based spatial analysis method (CTSA) for assessment of femoral hip implant migration: In vitro accuracy and precision comparable to that of radiostereometric analysis (RSA).

PubMed

Scheerlinck, Thierry; Polfliet, Mathias; Deklerck, Rudi; Van Gompel, Gert; Buls, Nico; Vandemeulebroucke, Jef

2016-01-01

We developed a marker-free automated CT-based spatial analysis (CTSA) method to detect stem-bone migration in consecutive CT datasets and assessed the accuracy and precision in vitro. Our aim was to demonstrate that in vitro accuracy and precision of CTSA is comparable to that of radiostereometric analysis (RSA). Stem and bone were segmented in 2 CT datasets and both were registered pairwise. The resulting rigid transformations were compared and transferred to an anatomically sound coordinate system, taking the stem as reference. This resulted in 3 translation parameters and 3 rotation parameters describing the relative amount of stem-bone displacement, and it allowed calculation of the point of maximal stem migration. Accuracy was evaluated in 39 comparisons by imposing known stem migration on a stem-bone model. Precision was estimated in 20 comparisons based on a zero-migration model, and in 5 patients without stem loosening. Limits of the 95% tolerance intervals (TIs) for accuracy did not exceed 0.28 mm for translations and 0.20° for rotations (largest standard deviation of the signed error (SD(SE)): 0.081 mm and 0.057°). In vitro, limits of the 95% TI for precision in a clinically relevant setting (8 comparisons) were below 0.09 mm and 0.14° (largest SD(SE): 0.012 mm and 0.020°). In patients, the precision was lower, but acceptable, and dependent on CT scan resolution. CTSA allows detection of stem-bone migration with an accuracy and precision comparable to that of RSA. It could be valuable for evaluation of subtle stem loosening in clinical practice.

Beneficial effect of Mentha suaveolens essential oil in the treatment of vaginal candidiasis assessed by real-time monitoring of infection

PubMed Central

2011-01-01

Background Vaginal candidiasis is a frequent and common distressing disease affecting up to 75% of the women of fertile age; most of these women have recurrent episodes. Essential oils from aromatic plants have been shown to have antimicrobial and antifungal activities. This study was aimed at assessing the anti-fungal activity of essential oil from Mentha suaveolens (EOMS) in an experimental infection of vaginal candidiasis. Methods The in vitro and in vivo activity of EOMS was assessed. The in vitro activity was evaluated under standard CLSI methods, and the in vivo analysis was carried out by exploiting a novel, non-invasive model of vaginal candidiasis in mice based on an in vivo imaging technique. Differences between essential oil treated and saline treated mice were evaluated by the non-parametric Mann-Whitney U-test. Viable count data from a time kill assay and yeast and hyphae survival test were compared using the Student's t-test (two-tailed). Results Our main findings were: i) EOMS shows potent candidastatic and candidacidal activity in an in vitro experimental system; ii) EOMS gives a degree of protection against vaginal candidiasis in an in vivo experimental system. Conclusions This study shows for the first time that the essential oil of a Moroccan plant Mentha suaveolens is candidastatic and candidacidal in vitro, and has a degree of anticandidal activity in a model of vaginal infection, as demonstrated in an in vivo monitoring imaging system. We conclude that our findings lay the ground for further, more extensive investigations to identify the active EOMS component(s), promising in the therapeutically problematic setting of chronic vaginal candidiasis in humans. PMID:21356078

Applying phasor approach analysis of multiphoton FLIM measurements to probe the metabolic activity of three-dimensional in vitro cell culture models

PubMed Central

Lakner, Pirmin H.; Monaghan, Michael G.; Möller, Yvonne; Olayioye, Monilola A.; Schenke-Layland, Katja

2017-01-01

Fluorescence lifetime imaging microscopy (FLIM) can measure and discriminate endogenous fluorophores present in biological samples. This study seeks to identify FLIM as a suitable method to non-invasively detect a shift in cellular metabolic activity towards glycolysis or oxidative phosphorylation in 3D Caco-2 models of colorectal carcinoma. These models were treated with potassium cyanide or hydrogen peroxide as controls, and epidermal growth factor (EGF) as a physiologically-relevant influencer of cell metabolic behaviour. Autofluorescence, attributed to nicotinamide adenine dinucleotide (NADH), was induced by two-photon laser excitation and its lifetime decay was analysed using a standard multi-exponential decay approach and also a novel custom-written code for phasor-based analysis. While both methods enabled detection of a statistically significant shift of metabolic activity towards glycolysis using potassium cyanide, and oxidative phosphorylation using hydrogen peroxide, employing the phasor approach required fewer initial assumptions to quantify the lifetimes of contributing fluorophores. 3D Caco-2 models treated with EGF had increased glucose consumption, production of lactate, and presence of ATP. FLIM analyses of these cultures revealed a significant shift in the contribution of protein-bound NADH towards free NADH, indicating increased glycolysis-mediated metabolic activity. This data demonstrate that FLIM is suitable to interpret metabolic changes in 3D in vitro models. PMID:28211922

Applying phasor approach analysis of multiphoton FLIM measurements to probe the metabolic activity of three-dimensional in vitro cell culture models.

PubMed

Lakner, Pirmin H; Monaghan, Michael G; Möller, Yvonne; Olayioye, Monilola A; Schenke-Layland, Katja

2017-02-13

Fluorescence lifetime imaging microscopy (FLIM) can measure and discriminate endogenous fluorophores present in biological samples. This study seeks to identify FLIM as a suitable method to non-invasively detect a shift in cellular metabolic activity towards glycolysis or oxidative phosphorylation in 3D Caco-2 models of colorectal carcinoma. These models were treated with potassium cyanide or hydrogen peroxide as controls, and epidermal growth factor (EGF) as a physiologically-relevant influencer of cell metabolic behaviour. Autofluorescence, attributed to nicotinamide adenine dinucleotide (NADH), was induced by two-photon laser excitation and its lifetime decay was analysed using a standard multi-exponential decay approach and also a novel custom-written code for phasor-based analysis. While both methods enabled detection of a statistically significant shift of metabolic activity towards glycolysis using potassium cyanide, and oxidative phosphorylation using hydrogen peroxide, employing the phasor approach required fewer initial assumptions to quantify the lifetimes of contributing fluorophores. 3D Caco-2 models treated with EGF had increased glucose consumption, production of lactate, and presence of ATP. FLIM analyses of these cultures revealed a significant shift in the contribution of protein-bound NADH towards free NADH, indicating increased glycolysis-mediated metabolic activity. This data demonstrate that FLIM is suitable to interpret metabolic changes in 3D in vitro models.

Butyrylation of Maize and Potato Starches and Characterization of the Products by Nuclear Magnetic Resonance and In Vitro Fermentation.

PubMed

Nielsen, Tina Skau; Canibe, Nuria; Larsen, Flemming Hofmann

2018-05-18

Intake of butyrylated starches may increase colonic butyrate supply, which can be of public health and clinical benefit by maintaining colonic health. The objective was to investigate if an organocatalytic method with tartaric acid as a catalyst could be applied to produce butyrylated products from different starch sources and to characterize their chemical structure and fermentation capability by using solid-state 13 C MAS NMR (magic angle spinning nuclear magnetic resonance) spectroscopy and an in vitro fermentation model, respectively. Low-amylose and high-amylose potato starch (LAPS and HAPS) and low-amylose and high-amylose maize starch (LAMS and HAMS) were subjected to organocatalytic butyrylation. This resulted in products with an increasing degree of substitution (DS) measured by heterogenous saponification and back titration with the HCl (chemical method) depending on reaction time. NMR analysis, however, showed that the major part of the acylation was induced by tartarate (75⁻89%) and only a minor part (11⁻25%) by butyrate. Generally, the chemical method overestimated the DS by 38% to 91% compared with the DS determination by NMR. Increasing the DS appeared to lower the in vitro fermentation capability of starches independent of the starch source and, therefore, do not seem to present a feasible method to deliver more butyrate to the colon than lower DS products.

Human Primary Intestinal Epithelial Cells as an Improved In Vitro Model for Cryptosporidium parvum Infection

PubMed Central

Cabada, Miguel M.; Nichols, Joan; Gomez, Guillermo; White, A. Clinton

2013-01-01

The study of human intestinal pathogens has been limited by the lack of methods for the long-term culture of primary human intestinal epithelial cells (PECs). The development of infection models with PECs would allow a better understanding of host-parasite interactions. The objective of this study was to develop a novel method for prolonged in vitro cultivation of PECs that can be used to study Cryptosporidium infection. We isolated intact crypts from human intestines removed during weight loss surgery. The fragments of intestinal layers were cultivated with culture medium supplemented with growth factors and antiapoptotic molecules. After 7 days, the PECs formed self-regenerating cell clusters, forming villi that resemble intestinal epithelium. The PECs proliferated and remained viable for at least 60 days. The cells expressed markers for intestinal stem cells, epithelial cells, and mature enterocytes. The PECs were infected with Cryptosporidium. In contrast to older models in which parasite numbers decay, the burden of parasites increased for >120 h. In summary, we describe here a novel method for the cultivation of self-regenerating human epithelial cells from small intestinal crypts, which contain both intestinal stem cells and mature villus cells. We present data that suggest these cells support Cryptosporidium better than existing cell lines. PECs should provide an improved tool for studying host-parasite interactions involving Cryptosporidium and other intestinal pathogens. PMID:23509153

In vitro blood-brain barrier permeability predictions for GABAA receptor modulating piperine analogs.

PubMed

Eigenmann, Daniela Elisabeth; Dürig, Carmen; Jähne, Evelyn Andrea; Smieško, Martin; Culot, Maxime; Gosselet, Fabien; Cecchelli, Romeo; Helms, Hans Christian Cederberg; Brodin, Birger; Wimmer, Laurin; Mihovilovic, Marko D; Hamburger, Matthias; Oufir, Mouhssin

2016-06-01

The alkaloid piperine from black pepper (Piper nigrum L.) and several synthetic piperine analogs were recently identified as positive allosteric modulators of γ-aminobutyric acid type A (GABAA) receptors. In order to reach their target sites of action, these compounds need to enter the brain by crossing the blood-brain barrier (BBB). We here evaluated piperine and five selected analogs (SCT-66, SCT-64, SCT-29, LAU397, and LAU399) regarding their BBB permeability. Data were obtained in three in vitro BBB models, namely a recently established human model with immortalized hBMEC cells, a human brain-like endothelial cells (BLEC) model, and a primary animal (bovine endothelial/rat astrocytes co-culture) model. For each compound, quantitative UHPLC-MS/MS methods in the range of 5.00-500ng/mL in the corresponding matrix were developed, and permeability coefficients in the three BBB models were determined. In vitro predictions from the two human BBB models were in good agreement, while permeability data from the animal model differed to some extent, possibly due to protein binding of the screened compounds. In all three BBB models, piperine and SCT-64 displayed the highest BBB permeation potential. This was corroborated by data from in silico prediction. For the other piperine analogs (SCT-66, SCT-29, LAU397, and LAU399), BBB permeability was low to moderate in the two human BBB models, and moderate to high in the animal BBB model. Efflux ratios (ER) calculated from bidirectional permeability experiments indicated that the compounds were likely not substrates of active efflux transporters. Copyright © 2016 Elsevier B.V. All rights reserved.

Exploration and comparison of in vitro eye irritation tests with the ISO standard in vivo rabbit test for the evaluation of the ocular irritancy of contact lenses.

PubMed

Yun, Jun-Won; Hailian, Quan; Na, Yirang; Kang, Byeong-Cheol; Yoon, Jung-Hee; Cho, Eun-Young; Lee, Miri; Kim, Da-Eun; Bae, SeungJin; Seok, Seung Hyeok; Lim, Kyung-Min

2016-12-01

In an effort to explore the use of alternative methods to animal testing for the evaluation of the ocular irritancy of medical devices, we evaluated representative contact lenses with the bovine corneal opacity and permeability test (BCOP) and an in vitro eye irritation test using the three-dimensionally-reconstructed human corneal epithelium (RhCE) models, EpiOcular™ and MCTT HCE™. In addition, we compared the obtained results with the ISO standard in vivo rabbit eye irritation test (ISO10993-10). Along with the positive controls (benzalkonium chloride, BAK, 0.02, 0.2, and 1%), the extracts of 4 representative contact lenses (soft, disposable, hard, and colored lenses) and 2 reference lenses (dye-eluting and BAK-coated lenses) were tested. All the lenses, except for the BAK-coated lens, were determined non-irritants in all test methods, while the positive controls yielded relevant results. More importantly, BCOP, EpiOcular™, and MCTT HCE™ yielded a consistent decision for all the tested samples, with the exception of 0.2% BAK in BCOP, for which no prediction could be made. Overall, all the in vitro tests correlated well with the in vivo rabbit eye irritation test, and furthermore, the combination of in vitro tests as a tiered testing strategy was able to produce results similar to those seen in vivo. These observations suggest that such methods can be used as alternative assays to replace the conventional in vivo test method in the evaluation of the ocular irritancy of ophthalmic medical devices, although further study is necessary. Copyright © 2016. Published by Elsevier Ltd.

In Vitro Interactions between Tacrolimus and Azoles against Candida albicans Determined by Different Methods▿

PubMed Central

Sun, Shujuan; Li, Yan; Guo, Qiongjie; Shi, Changwen; Yu, Jinlong; Ma, Lin

2008-01-01

Combination therapy could be of use for the treatment of fungal infections, especially those caused by drug-resistant fungi. However, the methods and approaches used for data generation and result interpretation need further optimizing. The fractional inhibitory concentration index (FICI) is the most commonly used method, but it has several drawbacks in characterizing antifungal drug interaction. Alternatively, some new methods can be used such as the ΔE model (difference between the predicted and measured fungal growth percentages) and the response surface approach, which uses the concentration-effect relationship over the whole concentration range instead of just the MIC. In the present study, in vitro interactions between tacrolimus (FK506) and three azoles—fluconazole (FLC), itraconazole (ITR), and voriconazole (VRC)-against Candida albicans were evaluated by the checkerboard microdilution method and time-killing test. The intensity of the interactions was determined by visual reading and the spectrophotometric method in a checkerboard assay, and the nature of the interactions was assessed by nonparametric models of FICI and ΔE. Colony counting and colorimetric viable detection methods (2,3-bis {2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium hydroxide} [XTT] reduction test) were used for evaluating the combination antifungal effects over time. Synergistic and indifferent effects were found for the combination of FK506 and azoles against azole-sensitive strains, while strong synergy was found against azole-resistant strains analyzed by FICI. The ΔE model gave more consistent results with FICI. The positive interactions were also confirmed by the time-killing test. Our findings suggest a potential role for combination therapy with calcineurin pathway inhibitors and azoles to augment activity against resistant C. albicans. PMID:18056277

A novel two-step hierarchical quantitative structure-activity relationship modeling work flow for predicting acute toxicity of chemicals in rodents.

PubMed

Zhu, Hao; Ye, Lin; Richard, Ann; Golbraikh, Alexander; Wright, Fred A; Rusyn, Ivan; Tropsha, Alexander

2009-08-01

Accurate prediction of in vivo toxicity from in vitro testing is a challenging problem. Large public-private consortia have been formed with the goal of improving chemical safety assessment by the means of high-throughput screening. A wealth of available biological data requires new computational approaches to link chemical structure, in vitro data, and potential adverse health effects. A database containing experimental cytotoxicity values for in vitro half-maximal inhibitory concentration (IC(50)) and in vivo rodent median lethal dose (LD(50)) for more than 300 chemicals was compiled by Zentralstelle zur Erfassung und Bewertung von Ersatz- und Ergaenzungsmethoden zum Tierversuch (ZEBET; National Center for Documentation and Evaluation of Alternative Methods to Animal Experiments). The application of conventional quantitative structure-activity relationship (QSAR) modeling approaches to predict mouse or rat acute LD(50) values from chemical descriptors of ZEBET compounds yielded no statistically significant models. The analysis of these data showed no significant correlation between IC(50) and LD(50). However, a linear IC(50) versus LD(50) correlation could be established for a fraction of compounds. To capitalize on this observation, we developed a novel two-step modeling approach as follows. First, all chemicals are partitioned into two groups based on the relationship between IC(50) and LD(50) values: One group comprises compounds with linear IC(50) versus LD(50) relationships, and another group comprises the remaining compounds. Second, we built conventional binary classification QSAR models to predict the group affiliation based on chemical descriptors only. Third, we developed k-nearest neighbor continuous QSAR models for each subclass to predict LD(50) values from chemical descriptors. All models were extensively validated using special protocols. The novelty of this modeling approach is that it uses the relationships between in vivo and in vitro data only to inform the initial construction of the hierarchical two-step QSAR models. Models resulting from this approach employ chemical descriptors only for external prediction of acute rodent toxicity.

More Realistic Face Model Surface Improves Relevance of Pediatric In-Vitro Aerosol Studies

PubMed Central

Amirav, Israel; Halamish, Asaf; Gorenberg, Miguel; Omar, Hamza; Newhouse, Michael T.

2015-01-01

Background Various hard face models are commonly used to evaluate the efficiency of aerosol face masks. Softer more realistic “face” surface materials, like skin, deform upon mask application and should provide more relevant in-vitro tests. Studies that simultaneously take into consideration many of the factors characteristic of the in vivo face are lacking. These include airways, various application forces, comparison of various devices, comparison with a hard-surface model and use of a more representative model face based on large numbers of actual faces. Aim To compare mask to “face” seal and aerosol delivery of two pediatric masks using a soft vs. a hard, appropriately representative, pediatric face model under various applied forces. Methods Two identical face models and upper airways replicas were constructed, the only difference being the suppleness and compressibility of the surface layer of the “face.” Integrity of the seal and aerosol delivery of two different masks [AeroChamber (AC) and SootherMask (SM)] were compared using a breath simulator, filter collection and realistic applied forces. Results The soft “face” significantly increased the delivery efficiency and the sealing characteristics of both masks. Aerosol delivery with the soft “face” was significantly greater for the SM compared to the AC (p< 0.01). No statistically significant difference between the two masks was observed with the hard “face.” Conclusions The material and pliability of the model “face” surface has a significant influence on both the seal and delivery efficiency of face masks. This finding should be taken into account during in-vitro aerosol studies. PMID:26090661

Specimen-specific modeling of hip fracture pattern and repair.

PubMed

Ali, Azhar A; Cristofolini, Luca; Schileo, Enrico; Hu, Haixiang; Taddei, Fulvia; Kim, Raymond H; Rullkoetter, Paul J; Laz, Peter J

2014-01-22

Hip fracture remains a major health problem for the elderly. Clinical studies have assessed fracture risk based on bone quality in the aging population and cadaveric testing has quantified bone strength and fracture loads. Prior modeling has primarily focused on quantifying the strain distribution in bone as an indicator of fracture risk. Recent advances in the extended finite element method (XFEM) enable prediction of the initiation and propagation of cracks without requiring a priori knowledge of the crack path. Accordingly, the objectives of this study were to predict femoral fracture in specimen-specific models using the XFEM approach, to perform one-to-one comparisons of predicted and in vitro fracture patterns, and to develop a framework to assess the mechanics and load transfer in the fractured femur when it is repaired with an osteosynthesis implant. Five specimen-specific femur models were developed from in vitro experiments under a simulated stance loading condition. Predicted fracture patterns closely matched the in vitro patterns; however, predictions of fracture load differed by approximately 50% due to sensitivity to local material properties. Specimen-specific intertrochanteric fractures were induced by subjecting the femur models to a sideways fall and repaired with a contemporary implant. Under a post-surgical stance loading, model-predicted load sharing between the implant and bone across the fracture surface varied from 59%:41% to 89%:11%, underscoring the importance of considering anatomic and fracture variability in the evaluation of implants. XFEM modeling shows potential as a macro-level analysis enabling fracture investigations of clinical cohorts, including at-risk groups, and the design of robust implants. © 2013 Published by Elsevier Ltd.

In vivo bioavailability and in vitro bioaccessibility of perfluorooctanoic acid (PFOA) in food matrices: correlation analysis and method development.

PubMed

Li, Kan; Li, Chao; Yu, Nan-Yang; Juhasz, Albert L; Cui, Xin-Yi; Ma, Lena Q

2015-01-06

Food is a major source of human exposure to perfluorooctanoic acid (PFOA), however, PFOA bioavailability in food has not been studied. An in vivo mouse model and three in vitro methods (unified BARGE method, UBM; physiologically based extraction test, PBET; and in vitro digestion method, IVD) were used to determine the relative bioavailability and bioaccessibility of PFOA in the presence of 17 foods. PFOA was mixed with foods of different nutritional compositions and fed to mice over a 7-d period. PFOA relative bioavailability was determined by comparing PFOA accumulation in the liver following PFOA exposure via food to that in water. PFOA bioavailability relative to water ranged from 4.30 ± 0.80 to 69.0 ± 11.9% and was negatively correlated with lipid content (r = 0.76). This was possibly due to competitive sorption of free fatty acids with PFOA onto transporters on intestine epithelial cells. Besides, cations in the gastrointestinal tract, such as Ca(2+) and Mg(2+), are capable of complexing PFOA and partitioning to the lipid phase. On the other hand, when assessed using in vitro assays, PFOA bioaccessibility varied with methods, being 8.7-73% (UBM), 9.8-99% (PBET), and 21-114% (IVD). PFOA bioaccessibility was negatively correlated with lipid content when assessed using UBM (r = 0.82); however, a poor correlation with food composition was observed for PBET and IVD (r = 0.01-0.50). When in vivo and in vitro data were compared, a strong correlation was observed for UBM (r = 0.79), but poor relationships were observed for PBET and IVD (r = 0.11-0.22). This was probably because the higher lipolysis ability and presence of Ca(2+) and Mg(2+) in the gastrointestinal fluid of UBM resulted in a lower potential to form stable micelles compared to PBET and IVD. These results indicated that PFOA relative bioavailability was mainly affected by lipid content in foods, and UBM has the potential to determine PFOA bioaccessibility in food samples.

Antioxidant Activity of Pistacia vera Fruits, Leaves and Gum Extracts

PubMed Central

Hosseinzadeh, Hossein; Sajadi Tabassi, Sayyed Abolghasem; Milani Moghadam, Negar; Rashedinia, Marzieh; Mehri, Soghra

2012-01-01

The side effects of synthetic antioxidants have been considered in different studies. Accordingly, there is an increasing interest toward the use of natural substances instead of the synthetic ones. In this study, the aqueous and ethanolic extracts of Pistacia vera leaves and fruits as well as hydroalcoholic extract of gum were tested for a possible antioxidant activity using in vitro methods. Deoxyribose assay, erythrocyte membrane lipid peroxidation and liver misrosomal non- enzymatic lipid peroxidation tests were used as an in-vitro model for determination antioxidant activity. The extract were evaluated at different concentratios: 25,100, 250, 500 and 1000 μg/mL. In all procedures, all extracts showed free radical scavenging activity. The effect of ethanolic extract of P. vera fruit at 1000 μg/mL was quite similar to positive control (DMSO 20 mM) in deoxyribose method. In two other tests, the ethanolic extracts of fruits and leaves were more effective than the aqueous extracts to inhibit malondialdehyde generation. Phytochemical tests showed the presence of flavonoids and tannins in Pistocia vera extracts. The present study showed that extracts of different part of P. vera have antioxidant activity in different in vitro methods. The ethanolic extracts of leaves and fruits showed more roles for antioxidant properties and gum hydroalcoholic extract demonstrated less antioxidant effect. PMID:24250515

A novel, simplified ex vivo method for measuring water exchange performance of heat and moisture exchangers for tracheostomy application.

PubMed

van den Boer, Cindy; Muller, Sara H; Vincent, Andrew D; Züchner, Klaus; van den Brekel, Michiel W M; Hilgers, Frans J M

2013-09-01

Breathing through a tracheostomy results in insufficient warming and humidification of inspired air. This loss of air-conditioning can be partially compensated for with the application of a heat and moisture exchanger (HME) over the tracheostomy. In vitro (International Organization for Standardization [ISO] standard 9360-2:2001) and in vivo measurements of the effects of an HME are complex and technically challenging. The aim of this study was to develop a simple method to measure the ex vivo HME performance comparable with previous in vitro and in vivo results. HMEs were weighed at the end of inspiration and at the end of expiration at different breathing volumes. Four HMEs (Atos Medical, Hörby, Sweden) with known in vivo humidity and in vitro water loss values were tested. The associations between weight change, volume, and absolute humidity were determined using both linear and non-linear mixed effects models. The rating between the 4 HMEs by weighing correlated with previous intra-tracheal measurements (R(2) = 0.98), and the ISO standard (R(2) = 0.77). Assessment of the weight change between end of inhalation and end of exhalation is a valid and simple method of measuring the water exchange performance of an HME.

Combination therapeutics of Nilotinib and radiation in acute lymphoblastic leukemia as an effective method against drug-resistance.

PubMed

Kaveh, Kamran; Takahashi, Yutaka; Farrar, Michael A; Storme, Guy; Guido, Marcucci; Piepenburg, Jamie; Penning, Jackson; Foo, Jasmine; Leder, Kevin Z; Hui, Susanta K

2017-07-01

Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) is characterized by a very poor prognosis and a high likelihood of acquired chemo-resistance. Although tyrosine kinase inhibitor (TKI) therapy has improved clinical outcome, most ALL patients relapse following treatment with TKI due to the development of resistance. We developed an in vitro model of Nilotinib-resistant Ph+ leukemia cells to investigate whether low dose radiation (LDR) in combination with TKI therapy overcome chemo-resistance. Additionally, we developed a mathematical model, parameterized by cell viability experiments under Nilotinib treatment and LDR, to explain the cellular response to combination therapy. The addition of LDR significantly reduced drug resistance both in vitro and in computational model. Decreased expression level of phosphorylated AKT suggests that the combination treatment plays an important role in overcoming resistance through the AKT pathway. Model-predicted cellular responses to the combined therapy provide good agreement with experimental results. Augmentation of LDR and Nilotinib therapy seems to be beneficial to control Ph+ leukemia resistance and the quantitative model can determine optimal dosing schedule to enhance the effectiveness of the combination therapy.

Neurons derived from different brain regions are inherently different in vitro: a novel multiregional brain-on-a-chip.

PubMed

Dauth, Stephanie; Maoz, Ben M; Sheehy, Sean P; Hemphill, Matthew A; Murty, Tara; Macedonia, Mary Kate; Greer, Angie M; Budnik, Bogdan; Parker, Kevin Kit

2017-03-01

Brain in vitro models are critically important to developing our understanding of basic nervous system cellular physiology, potential neurotoxic effects of chemicals, and specific cellular mechanisms of many disease states. In this study, we sought to address key shortcomings of current brain in vitro models: the scarcity of comparative data for cells originating from distinct brain regions and the lack of multiregional brain in vitro models. We demonstrated that rat neurons from different brain regions exhibit unique profiles regarding their cell composition, protein expression, metabolism, and electrical activity in vitro. In vivo, the brain is unique in its structural and functional organization, and the interactions and communication between different brain areas are essential components of proper brain function. This fact and the observation that neurons from different areas of the brain exhibit unique behaviors in vitro underline the importance of establishing multiregional brain in vitro models. Therefore, we here developed a multiregional brain-on-a-chip and observed a reduction of overall firing activity, as well as altered amounts of astrocytes and specific neuronal cell types compared with separately cultured neurons. Furthermore, this multiregional model was used to study the effects of phencyclidine, a drug known to induce schizophrenia-like symptoms in vivo, on individual brain areas separately while monitoring downstream effects on interconnected regions. Overall, this work provides a comparison of cells from different brain regions in vitro and introduces a multiregional brain-on-a-chip that enables the development of unique disease models incorporating essential in vivo features. NEW & NOTEWORTHY Due to the scarcity of comparative data for cells from different brain regions in vitro, we demonstrated that neurons isolated from distinct brain areas exhibit unique behaviors in vitro. Moreover, in vivo proper brain function is dependent on the connection and communication of several brain regions, underlining the importance of developing multiregional brain in vitro models. We introduced a novel brain-on-a-chip model, implementing essential in vivo features, such as different brain areas and their functional connections. Copyright © 2017 the American Physiological Society.

Neurons derived from different brain regions are inherently different in vitro: a novel multiregional brain-on-a-chip

PubMed Central

Dauth, Stephanie; Maoz, Ben M.; Sheehy, Sean P.; Hemphill, Matthew A.; Murty, Tara; Macedonia, Mary Kate; Greer, Angie M.; Budnik, Bogdan

2017-01-01

Brain in vitro models are critically important to developing our understanding of basic nervous system cellular physiology, potential neurotoxic effects of chemicals, and specific cellular mechanisms of many disease states. In this study, we sought to address key shortcomings of current brain in vitro models: the scarcity of comparative data for cells originating from distinct brain regions and the lack of multiregional brain in vitro models. We demonstrated that rat neurons from different brain regions exhibit unique profiles regarding their cell composition, protein expression, metabolism, and electrical activity in vitro. In vivo, the brain is unique in its structural and functional organization, and the interactions and communication between different brain areas are essential components of proper brain function. This fact and the observation that neurons from different areas of the brain exhibit unique behaviors in vitro underline the importance of establishing multiregional brain in vitro models. Therefore, we here developed a multiregional brain-on-a-chip and observed a reduction of overall firing activity, as well as altered amounts of astrocytes and specific neuronal cell types compared with separately cultured neurons. Furthermore, this multiregional model was used to study the effects of phencyclidine, a drug known to induce schizophrenia-like symptoms in vivo, on individual brain areas separately while monitoring downstream effects on interconnected regions. Overall, this work provides a comparison of cells from different brain regions in vitro and introduces a multiregional brain-on-a-chip that enables the development of unique disease models incorporating essential in vivo features. NEW & NOTEWORTHY Due to the scarcity of comparative data for cells from different brain regions in vitro, we demonstrated that neurons isolated from distinct brain areas exhibit unique behaviors in vitro. Moreover, in vivo proper brain function is dependent on the connection and communication of several brain regions, underlining the importance of developing multiregional brain in vitro models. We introduced a novel brain-on-a-chip model, implementing essential in vivo features, such as different brain areas and their functional connections. PMID:28031399

Development of an in vitro Hepatocyte Model to Investigate Chemical Mode of Action

EPA Science Inventory

There is a clear need to identify and characterize the potential of liver in vitro models that can be used to replace animals for mode of action analysis. Our goal is to use in vitro models for mode of action prediction which recapitulate critical cellular processes underlying in...

Molecular modeling of drug-pathophysiological Mtb protein targets: Synthesis of some 2-thioxo-1, 3-thiazolidin-4-one derivatives as anti-tubercular agents

NASA Astrophysics Data System (ADS)

Noorulla, K. M.; Suresh, Ayyadurai Jerad; Devaraji, Vinod; Mathew, Bijo; Umesh, Devi

2017-11-01

Twenty novel 2-thioxo-1, 3-thiazolidin-4-one derivatives (5a-5t) were synthesized and evaluated for their antitubercular activity. The structure of the compounds was confirmed by IR, NMR and Mass Spectroscopy methods. In addition, single-crystal X-ray diffraction was performed for compound 5a. All the synthesized compounds were screened for their in-vitro antimycobacterial activity against MTB (H37RV, ATCC No: 27294) by Alamar Blue assay method. Compounds 5r, 5k, 5t displayed most potent in-vitro activity with MICs of 0.05, 0.1, 0.2 μg/ml concentrations respectively which are comparatively potent than the standards. Molecular docking and dynamics simulations were performed to find out the plausible mechanism of the titled compounds.

Protective effect of NSA on intestinal epithelial cells in a necroptosis model

PubMed Central

Dong, Wei; Zhang, Min; Zhu, Yaxi; Chen, Yuanhan; Zhao, Xingchen; Li, Ruizhao; Zhang, Li; Ye, Zhiming; Liang, Xingling

2017-01-01

Objective This study aimed to investigate the protective effect of the necroptosis inhibitor necrosulfonamide (NSA) on intestinal epithelial cells using a novel in vitro necroptosis model that mimics inflammatory bowel disease (IBD). Methods 2,4,6-trinitrobenzenesulfonic acid (TNBS) was perfused into the rectum of BALB/c mice to established a colitis model. Pathologic injury and cell death were evaluated. A novel in vitro model of necroptosis was established in Caco-2 cells using TNF-α and Z-VAD-fmk, and the cells were treated with or without NSA. Morphologic changes, manner of cell death and the levels of phosphorylation of receptor-interacting protein kinase 3 (p-RIPK3) and mixed-lineage kinase domain-like (p-MLKL) were detected. Results In the TNBS-induced colitis in mice, TUNEL-positive and caspase-3-negative cells were observed in the intestinal mucosa, and p-RIPK3 was found to be elevated. Under the stimulation of TNF-α and Z-VAD-fmk, the morphologic damage in the Caco-2 cells was aggravated, the proportion of necrosis was increased, and the level of p-RIPK3 and p-MLKL were increased, confirming that the regulated cell death was necroptosis. NSA reversed the morphological abnormalities and reduced necrotic cell death induced by TNF-α and Z-VAD-fmk. Conclusion NSA can inhibit necroptosis in intestinal epithelial cells in vitro and might confer a potential protective effect against IBD. PMID:29156831

Evaluation of Microelectrode Array Data using Bayesian Modeling as an Approach to Screening and Prioritization for Neurotoxicity Testing*

EPA Science Inventory

The need to assess large numbers of chemicals for their potential toxicities has resulted in increased emphasis on medium- and high-throughput in vitro screening approaches. For such approaches to be useful, efficient and reliable data analysis and hit detection methods are also ...

Application of the ToxMiner Database: Network Analysis Linking the ToxCast Chemicals to Known Disease-Gene Associations

EPA Science Inventory

The US EPA ToxCast program is using in vitro HTS (High-Throughput Screening) methods to profile and model bioactivity of environmental chemicals. The main goals of the ToxCast program are to generate predictive signatures of toxicity, and ultimately provide rapid and cost-effecti...

A Novel Two-Step Hierarchial Quantitative Structure-Activity Relationship Modeling Workflow for Predicting Acute Toxicity of Chemicals in Rodents

EPA Science Inventory

Background: Accurate prediction of in vivo toxicity from in vitro testing is a challenging problem. Large public–private consortia have been formed with the goal of improving chemical safety assessment by the means of high-throughput screening. Methods and results: A database co...

ASSESSMENT OF CHEMICAL EFFECTS ON NEURONAL DIFFERENTIATION USING THE ARRAYSCAN HIGH CONTENT SCREENING SYSTEM

EPA Science Inventory

The development of alternative methods for toxicity testing is driven by the need for scientifically valid data that can be obtained in a rapid and cost-efficient manner. In vitro systems provide a model in which chemical effects on cellular events can be examined using technique...

Informatics approach using metabolic reactivity classifiers to link in vitro to in vivo data in application to the ToxCast Phase I dataset

EPA Science Inventory

Strategic combinations and tiered application of alternative testing methods to replace or minimize the use of animal models is attracting much attention. With the advancement of high throughput screening (HTS) assays and legacy databases providing in vivo testing results, suffic...

Development of a High-Content Image Analysis Method for Quantifying Synaptic Contacts in Rodent Primary Neuronal Cultures

EPA Science Inventory

Development of the nervous system occurs through a series of critical processes, each of which may be sensitive to disruption by environmental contaminants. In vitro culture of neurons can be used to model these processes and evaluate the potential of chemicals to act as develop...

[Effects of electromagnetic pulse exposure on the permeability of inner blood-retinal barrier model in vitro].

PubMed

Li, Hai-juan; Yang, Long-long; Tian, Wei; Liu, Jun-ju; Xie, Xue-jun; Guo, Guo-zhen

2012-03-01

To establish the inner blood-retinal barrier (BRB) model in vitro by co-culturing RF/6A cells and C6 cells and to investigate the effects of EMP (200 kV/m, 200 pulses) exposure on the permeability of the inner BRB model in vitro. RF/6A cells and C6 cells were co-cultured on transwell, and the characteristic of the inner BRB model was assessed by detecting transendothelial electrical resistance (TEER) and the permeability of horseradish peroxidase (HRP). The co-cultured model was exposed or sham exposed to the EMP (200 kV/m 200 pulses) for 0.5, 3, 6, 12, 24 h in vitro, then TEER and the permeability of HRP were measured for studying the effects of EMP on the permeability of inner BRB model in vitro. TEER value (145 Ωcm(2)) of the co-culturing inner BRB model significantly increased, as compared to that of RF/6A cells alone model (P < 0.05) on the 6th day after inoculation. There was significant difference of permeability of HRP between the co-culturing inner BRB model and RF/6A cells alone model (P < 0.05). The ability of inhibiting large molecular materials in the co-culturing inner BRB model enhanced. The TEER value decreased and the permeability of HRP increased as compared to the sham group at 0.5, 3, 6 h after the exposure. The inner BRB model by co-culturing RF/6A cells and C6 cells in vitro is efficient and suitable to study the alterations of the restricted permeability function of the inner BRB. EMP (200 kV/m for 200 pulses) could induce the enhanced permeability of the inner BRB model in vitro.

The identification of protein domains that mediate functional interactions between Rab-GTPases and RabGAPs using 3D protein modeling.

PubMed

Davie, Jeremiah J; Faitar, Silviu L

2017-01-01

Currently, time-consuming serial in vitro experimentation involving immunocytochemistry or radiolabeled materials is required to identify which of the numerous Rab-GTPases (Rab) and Rab-GTPase activating proteins (RabGAP) are capable of functional interactions. These interactions are essential for numerous cellular functions, and in silico methods of reducing in vitro trial and error would accelerate the pace of research in cell biology. We have utilized a combination of three-dimensional protein modeling and protein bioinformatics to identify domains present in Rab proteins that are predictive of their functional interaction with a specific RabGAP. The RabF2 and RabSF1 domains appear to play functional roles in mediating the interaction between Rabs and RabGAPs. Moreover, the RabSF1 domain can be used to make in silico predictions of functional Rab/RabGAP pairs. This method is expected to be a broadly applicable tool for predicting protein-protein interactions where existing crystal structures for homologs of the proteins of interest are available.

Comparison of methods for the prediction of human clearance from hepatocyte intrinsic clearance for a set of reference compounds and an external evaluation set.

PubMed

Yamagata, Tetsuo; Zanelli, Ugo; Gallemann, Dieter; Perrin, Dominique; Dolgos, Hugues; Petersson, Carl

2017-09-01

1. We compared direct scaling, regression model equation and the so-called "Poulin et al." methods to scale clearance (CL) from in vitro intrinsic clearance (CL int ) measured in human hepatocytes using two sets of compounds. One reference set comprised of 20 compounds with known elimination pathways and one external evaluation set based on 17 compounds development in Merck (MS). 2. A 90% prospective confidence interval was calculated using the reference set. This interval was found relevant for the regression equation method. The three outliers identified were justified on the basis of their elimination mechanism. 3. The direct scaling method showed a systematic underestimation of clearance in both the reference and evaluation sets. The "Poulin et al." and the regression equation methods showed no obvious bias in either the reference or evaluation sets. 4. The regression model equation was slightly superior to the "Poulin et al." method in the reference set and showed a better absolute average fold error (AAFE) of value 1.3 compared to 1.6. A larger difference was observed in the evaluation set were the regression method and "Poulin et al." resulted in an AAFE of 1.7 and 2.6, respectively (removing the three compounds with known issues mentioned above). A similar pattern was observed for the correlation coefficient. Based on these data we suggest the regression equation method combined with a prospective confidence interval as the first choice for the extrapolation of human in vivo hepatic metabolic clearance from in vitro systems.

Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro.

PubMed

Knight, Eleanor; Przyborski, Stefan

2015-12-01

Research in mammalian cell biology often relies on developing in vitro models to enable the growth of cells in the laboratory to investigate a specific biological mechanism or process under different test conditions. The quality of such models and how they represent the behavior of cells in real tissues plays a critical role in the value of the data produced and how it is used. It is particularly important to recognize how the structure of a cell influences its function and how co-culture models can be used to more closely represent the structure of real tissue. In recent years, technologies have been developed to enhance the way in which researchers can grow cells and more readily create tissue-like structures. Here we identify the limitations of culturing mammalian cells by conventional methods on two-dimensional (2D) substrates and review the popular approaches currently available that enable the development of three-dimensional (3D) tissue models in vitro. There are now many ways in which the growth environment for cultured cells can be altered to encourage 3D cell growth. Approaches to 3D culture can be broadly categorized into scaffold-free or scaffold-based culture systems, with scaffolds made from either natural or synthetic materials. There is no one particular solution that currently satisfies all requirements and researchers must select the appropriate method in line with their needs. Using such technology in conjunction with other modern resources in cell biology (e.g. human stem cells) will provide new opportunities to create robust human tissue mimetics for use in basic research and drug discovery. Application of such models will contribute to advancing basic research, increasing the predictive accuracy of compounds, and reducing animal usage in biomedical science. © 2014 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.

An in-vitro-in-vivo model for the transdermal delivery of cholecalciferol for the purposes of rodent management.

PubMed

Davies, J; Ingham, A

2015-06-20

The natural selection of anticoagulant resistant rats has resulted in a need for an alternative to anticoagulant rodenticides which differs in both active ingredient and in the method of dosing. Cholecalciferol toxicity to rodents using the dermal route is demonstrated using a variety of penetration enhancing formulations in two in-vitro models and finally in-vivo. A 1 ml dose of 50/50 (v/v) DMSO/ethanol containing 15% (v/v) PEG 200 and 20% (w/v) cholecalciferol was judged as 'sufficiently effective' in line with the European Union's Biocidal Products Regulation (No. 528/2012) during in-vivo studies. This dose was found to cause 100% mortality in a rat population in 64.4h (± 22h). Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Toxcast and the Use of Human Relevant In Vitro Exposures ...

EPA Pesticide Factsheets

The path for incorporating new approach methods and technologies into quantitative chemical risk assessment poses a diverse set of scientific challenges. These challenges include sufficient coverage of toxicological mechanisms to meaningfully interpret negative test results, development of increasingly relevant test systems, computational modeling to integrate experimental data, putting results in a dose and exposure context, characterizing uncertainty, and efficient validation of the test systems and computational models. The presentation will cover progress at the U.S. EPA in systematically addressing each of these challenges and delivering more human-relevant risk-based assessments. This abstract does not necessarily reflect U.S. EPA policy. Presentation at the British Toxicological Society Annual Congress on ToxCast and the Use of Human Relevant In Vitro Exposures: Incorporating high-throughput exposure and toxicity testing data for 21st century risk assessments .

Assessment of relative bioavailability of heavy metals in soil using in vivo mouse model and its implication for risk assessment compared with bioaccessibility using in vitro assay.

PubMed

Kang, Yuan; Pan, Weijian; Liang, Siyun; Li, Ning; Zeng, Lixuan; Zhang, Qiuyun; Luo, Jiwen

2016-10-01

There is limited study to simultaneously determine the relative bioavailability of heavy metals such as Cd, Pb, Cu, Cr(VI), and Ni in soil samples. In the present study, the bioaccessibility of heavy metals using in vitro assay was compared with the relative bioavailability of heavy metals using in vivo mouse model. The bioaccessibility of heavy metals ranged from 9.05 ± 0.97 % (Cr) to 42.8 ± 3.52 % (Cd). The uptake profile of heavy metals in soil and solution samples in mouse revealed that the uptake kinetics could be fitted to a two-compartment model. The relative bioavailability of heavy meals ranged from 34.8 ± 7.0 % (Ni) to 131 ± 20.3 % (Cu). Poor correlation between bioaccessibility and relative bioavailability of heavy metals was observed (r (2) = 0.11, p > 0.05). The relative bioavailability of heavy metals was significantly higher than the bioaccessibility of heavy metals (p < 0.05). The present study indicated that the in vitro digestion method should be carefully employed in risk assessment.

Design and in vivo evaluation of carvedilol buccal mucoadhesive patches.

PubMed

Thimmasetty, J; Pandey, G S; Babu, P R Sathesh

2008-07-01

The buccal region offers an attractive route of administration for systemic drug delivery. Carvedilol (dose, 3.125-25 mg) is beta-adrenergic antagonist. Its oral bioavailability is 25-35% because of first pass metabolism. Buccal absorption studies of a carvedilol solution in human volunteers showed 32.86% drug absorption. FTIR and UV spectroscopic methods revealed that there was no interaction between carvedilol and polymers. Carvedilol patches were prepared using HPMC, carbopol 934, eudragit RS 100, and ethylcellulose. The patches were evaluated for their thickness uniformity, folding endurance, weight uniformity, content uniformity, swelling behaviour, tensile strength, and surface pH. In vitro release studies were conducted for carvedilol-loaded patches in phosphate buffer (pH, 6.6) solution. Patches exhibited drug release in the range of 86.26 to 98.32% in 90 min. Data of in vitro release from patches were fit to different equations and kinetic models to explain release profiles. Kinetic models used were zero and first-order equations, Hixon-Crowell, Higuchi, and Korsmeyer-Peppas models. In vivo drug release studies in rabbits showed 90.85% of drug release from HPMC-carbopol patch while it was 74.63 to 88.02% within 90 min in human volunteers. Good correlation among in vitro release and in vivo release of carvedilol was observed.

An in vitro lung model to assess true shunt fraction by multiple inert gas elimination.

PubMed

Varadarajan, Balamurugan; Vogt, Andreas; Hartwich, Volker; Vasireddy, Rakesh; Consiglio, Jolanda; Hugi-Mayr, Beate; Eberle, Balthasar

2017-01-01

The Multiple Inert Gas Elimination Technique, based on Micropore Membrane Inlet Mass Spectrometry, (MMIMS-MIGET) has been designed as a rapid and direct method to assess the full range of ventilation-to-perfusion (V/Q) ratios. MMIMS-MIGET distributions have not been assessed in an experimental setup with predefined V/Q-distributions. We aimed (I) to construct a novel in vitro lung model (IVLM) for the simulation of predefined V/Q distributions with five gas exchange compartments and (II) to correlate shunt fractions derived from MMIMS-MIGET with preset reference shunt values of the IVLM. Five hollow-fiber membrane oxygenators switched in parallel within a closed extracorporeal oxygenation circuit were ventilated with sweep gas (V) and perfused with human red cell suspension or saline (Q). Inert gas solution was infused into the perfusion circuit of the gas exchange assembly. Sweep gas flow (V) was kept constant and reference shunt fractions (IVLM-S) were established by bypassing one or more oxygenators with perfusate flow (Q). The derived shunt fractions (MM-S) were determined using MIGET by MMIMS from the retention data. Shunt derived by MMIMS-MIGET correlated well with preset reference shunt fractions. The in vitro lung model is a convenient system for the setup of predefined true shunt fractions in validation of MMIMS-MIGET.

[Evaluation on a fast weight reduction model in vitro].

PubMed

Li, Songtao; Li, Ying; Wen, Ying; Sun, Changhao

2010-03-01

To establish a fast and effective model in vitro for screening weight-reducing drugs and taking preliminary evaluation of the model. Mature adipocytes of SD rat induced by oleic acid were used to establish a obesity model in vitro. Isoprel, genistein, caffeine were selected as positive agents and curcumine as negative agent to evaluate the obesity model. Lipolysis of adipocytes was stimulated significantly by isoprel, genistein and caffeine rather than curcumine. This model could be used efficiently for screening weight-losing drugs.

Er:YAG laser-induced changes in skin in vivo and transdermal drug delivery

NASA Astrophysics Data System (ADS)

Flock, Stephen T.; Stern, Tom; Lehman, Paul; Dinehart, Scott; Franz, Tom; Liu, George; Stern, Scott J.

1997-05-01

It has been shown that laser ablation of stratum corneum, in vitro, can result in an increased uptake of topically applied pharmaceuticals. We have performed measurements of drug permeation, using an in vitro model of human skin, that involves a portable Er:YAG laser used to ablate the stratum corneum. For the first time, this method of drug administration was tested in vivo in human volunteers, whereby a hydrocortisone blanching assay was used to assess the efficiency of the procedure. The results show that this is a safe and efficient way to ablate stratum corneum for the purpose of enhanced transcutaneous drug administration.

Optimizing Culture Medium Composition to Improve Oligodendrocyte Progenitor Cell Yields In Vitro from Subventricular Zone-Derived Neural Progenitor Cell Neurospheres

PubMed Central

Franco, Paula G.; Pasquini, Juana M.; Silvestroff, Lucas

2015-01-01

Neural Stem and Progenitor Cells (NSC/NPC) are gathering tangible recognition for their uses in cell therapy and cell replacement therapies for human disease, as well as a model system to continue research on overall neural developmental processes in vitro. The Subventricular Zone is one of the largest NSC/NPC niches in the developing mammalian Central Nervous System, and persists through to adulthood. Oligodendrocyte progenitor cell (OPC) enriched cultures are usefull tools for in vitro studies as well as for cell replacement therapies for treating demyelination diseases. We used Subventricular Zone-derived NSC/NPC primary cultures from newborn mice and compared the effects of different growth factor combinations on cell proliferation and OPC yield. The Platelet Derived Growth Factor-AA and BB homodimers had a positive and significant impact on OPC generation. Furthermore, heparin addition to the culture media contributed to further increase overall culture yields. The OPC generated by this protocol were able to mature into Myelin Basic Protein-expressing cells and to interact with neurons in an in vitro co-culture system. As a whole, we describe an optimized in vitro method for increasing OPC. PMID:25837625

Compartmental modelling of the pharmacokinetics of a breast cancer resistance protein.

PubMed

Grandjean, Thomas R B; Chappell, Mike J; Yates, James T W; Jones, Kevin; Wood, Gemma; Coleman, Tanya

2011-11-01

A mathematical model for the pharmacokinetics of Hoechst 33342 following administration into a culture medium containing a population of transfected cells (HEK293 hBCRP) with a potent breast cancer resistance protein inhibitor, Fumitremorgin C (FTC), present is described. FTC is reported to almost completely annul resistance mediated by BCRP in vitro. This non-linear compartmental model has seven macroscopic sub-units, with 14 rate parameters. It describes the relationship between the concentration of Hoechst 33342 and FTC, initially spiked in the medium, and the observed change in fluorescence due to Hoechst 33342 binding to DNA. Structural identifiability analysis has been performed using two methods, one based on the similarity transformation/exhaustive modelling approach and the other based on the differential algebra approach. The analyses demonstrated that all models derived are uniquely identifiable for the experiments/observations available. A kinetic modelling software package, namely FACSIMILE (MPCA Software, UK), was used for parameter fitting and to obtain numerical solutions for the system equations. Model fits gave very good agreement with in vitro data provided by AstraZeneca across a variety of experimental scenarios. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Drug drug interaction extraction from the literature using a recursive neural network

PubMed Central

Lim, Sangrak; Lee, Kyubum

2018-01-01

Detecting drug-drug interactions (DDI) is important because information on DDIs can help prevent adverse effects from drug combinations. Since there are many new DDI-related papers published in the biomedical domain, manually extracting DDI information from the literature is a laborious task. However, text mining can be used to find DDIs in the biomedical literature. Among the recently developed neural networks, we use a Recursive Neural Network to improve the performance of DDI extraction. Our recursive neural network model uses a position feature, a subtree containment feature, and an ensemble method to improve the performance of DDI extraction. Compared with the state-of-the-art models, the DDI detection and type classifiers of our model performed 4.4% and 2.8% better, respectively, on the DDIExtraction Challenge’13 test data. We also validated our model on the PK DDI corpus that consists of two types of DDIs data: in vivo DDI and in vitro DDI. Compared with the existing model, our detection classifier performed 2.3% and 6.7% better on in vivo and in vitro data respectively. The results of our validation demonstrate that our model can automatically extract DDIs better than existing models. PMID:29373599

Agent-Based Computational Modeling of Cell Culture: Understanding Dosimetry In Vitro as Part of In Vitro to In Vivo Extrapolation

EPA Science Inventory

Quantitative characterization of cellular dose in vitro is needed for alignment of doses in vitro and in vivo. We used the agent-based software, CompuCell3D (CC3D), to provide a stochastic description of cell growth in culture. The model was configured so that isolated cells assu...

Characteristics of Aspergillus fumigatus in Association with Stenotrophomonas maltophilia in an In Vitro Model of Mixed Biofilm

PubMed Central

Melloul, Elise; Luiggi, Stéphanie; Anaïs, Leslie; Arné, Pascal; Costa, Jean-Marc; Fihman, Vincent; Briard, Benoit; Dannaoui, Eric; Guillot, Jacques; Decousser, Jean-Winoc; Beauvais, Anne; Botterel, Françoise

2016-01-01

Background Biofilms are communal structures of microorganisms that have long been associated with a variety of persistent infections poorly responding to conventional antibiotic or antifungal therapy. Aspergillus fumigatus fungus and Stenotrophomonas maltophilia bacteria are examples of the microorganisms that can coexist to form a biofilm especially in the respiratory tract of immunocompromised patients or cystic fibrosis patients. The aim of the present study was to develop and assess an in vitro model of a mixed biofilm associating S. maltophilia and A. fumigatus by using analytical and quantitative approaches. Materials and Methods An A. fumigatus strain (ATCC 13073) expressing a Green Fluorescent Protein (GFP) and an S. maltophilia strain (ATCC 13637) were used. Fungal and bacterial inocula (105 conidia/mL and 106 cells/mL, respectively) were simultaneously deposited to initiate the development of an in vitro mixed biofilm on polystyrene supports at 37°C for 24 h. The structure of the biofilm was analysed via qualitative microscopic techniques like scanning electron and transmission electron microscopy, and fluorescence microscopy, and by quantitative techniques including qPCR and crystal violet staining. Results Analytic methods revealed typical structures of biofilm with production of an extracellular matrix (ECM) enclosing fungal hyphae and bacteria. Quantitative methods showed a decrease of A. fumigatus growth and ECM production in the mixed biofilm with antibiosis effect of the bacteria on the fungi seen as abortive hyphae, limited hyphal growth, fewer conidia, and thicker fungal cell walls. Conclusion For the first time, a mixed A. fumigatus—S. maltophilia biofilm was validated by various analytical and quantitative approaches and the bacterial antibiosis effect on the fungus was demonstrated. The mixed biofilm model is an interesting experimentation field to evaluate efficiency of antimicrobial agents and to analyse the interactions between the biofilm and the airways epithelium. PMID:27870863

Comparison of 3D quantitative structure-activity relationship methods: Analysis of the in vitro antimalarial activity of 154 artemisinin analogues by hypothetical active-site lattice and comparative molecular field analysis

NASA Astrophysics Data System (ADS)

Woolfrey, John R.; Avery, Mitchell A.; Doweyko, Arthur M.

1998-03-01

Two three-dimensional quantitative structure-activity relationship (3D-QSAR) methods, comparative molecular field analysis (CoMFA) and hypothetical active site lattice (HASL), were compared with respect to the analysis of a training set of 154 artemisinin analogues. Five models were created, including a complete HASL and two trimmed versions, as well as two CoMFA models (leave-one-out standard CoMFA and the guided-region selection protocol). Similar r2 and q2 values were obtained by each method, although some striking differences existed between CoMFA contour maps and the HASL output. Each of the four predictive models exhibited a similar ability to predict the activity of a test set of 23 artemisinin analogues, although some differences were noted as to which compounds were described well by either model.

Development of an in vitro cell culture model to study milk to plasma ratios of therapeutic drugs.

PubMed

Athavale, Maithili A; Maitra, Anurupa; Patel, Shahnaz; Bhate, Vijay R; Toddywalla, Villi S

2013-01-01

To create an in vitro cell culture model to predict the M/P (concentration of drug in milk/concentration in maternal plasma) ratios of therapeutic drugs viz. rifampicin, theophylline, paracetamol, and aspirin. An in vitro cell culture model using CIT3 cells (mouse mammary epithelial cells) was created by culturing the cells on transwells. The cells formed an integral monolayer, allowing only transcellular transport as it happens in vivo. Functionality of the cells was confirmed through scanning electron microscopy. Time wise transfer of the study drugs from plasma to milk was studied and compared with actual (in vivo) M/P ratios obtained at reported tmax for the respective drugs. The developed model mimicked two important intrinsic factors of mammary epithelial cells viz. secretory and tight-junction properties and also the passive route of drug transport. The in vitro M/P ratios at reported tmax were 0.23, 0.61, 0.87, and 0.03 respectively, for rifampicin, theophylline, paracetamol, and salicylic acid as compared to 0.29, 0.65, 0.65, and 0.22, respectively, in vitro. Our preliminary effort to develop an in vitro physiological model showed promising results. Transfer rate of the drugs using the developed model compared well with the transfer potential seen in vivo except for salicylic acid, which was transferred in far lower concentration in vitro. The model has a potential to be developed as a non-invasive alternative to the in vitro technique for determining the transfer of therapeutic drugs into breast milk.

IN VITRO SCREENING BATTERIES FOR NEUROTOXICANTS

EPA Science Inventory

The need to develop, validate and utilize in vitro models to test chemicals for neurotoxic potential is widely appreciated. his lecture discusses the major advantages and disadvantages of using cell and tissue culture, the carious in vitro models amenable for neurotoxicity studie...

Lentiviral vector-mediated genetic modification of human neural progenitor cells for ex vivo gene therapy.

PubMed

Capowski, Elizabeth E; Schneider, Bernard L; Ebert, Allison D; Seehus, Corey R; Szulc, Jolanta; Zufferey, Romain; Aebischer, Patrick; Svendsen, Clive N

2007-07-30

Human neural progenitor cells (hNPC) hold great potential as an ex vivo system for delivery of therapeutic proteins to the central nervous system. When cultured as aggregates, termed neurospheres, hNPC are capable of significant in vitro expansion. In the current study, we present a robust method for lentiviral vector-mediated gene delivery into hNPC that maintains the differentiation and proliferative properties of neurosphere cultures while minimizing the amount of viral vector used and controlling the number of insertion sites per population. This method results in long-term, stable expression even after differentiation of the hNPC to neurons and astrocytes and allows for generation of equivalent transgenic populations of hNPC. In addition, the in vitro analysis presented predicts the behavior of transgenic lines in vivo when transplanted into a rodent model of Parkinson's disease. The methods presented provide a powerful tool for assessing the impact of factors such as promoter systems or different transgenes on the therapeutic utility of these cells.

The synergy of honokiol and fluconazole against clinical isolates of azole-resistant Candida albicans.

PubMed

Jin, J; Guo, N; Zhang, J; Ding, Y; Tang, X; Liang, J; Li, L; Deng, X; Yu, L

2010-09-01

To evaluate the interaction of fluconazole (FLC) and honokiol (HNK) in vitro and vivo against azole-resistant (azole-R) clinical isolates of Candida albicans. A checkerboard microdilution method was used to study the in vitro interaction of FLC and HNK in 24 azole-R clinical isolates of C. albicans. In vivo antifungal activity was performed to further analyse the interaction between FLC and HNK. In the in vitro study, synergism was observed in all 24 FLC-resistant strains tested as determined by fractional inhibitory concentration index (FICI), and in 22 strains by Delta E models. No antagonistic activity was observed in any of the strains tested. These positive interactions were also confirmed by using the time-killing test for the selected strain C. albicans YL371, which shows strong susceptible to the combination of HNK and FLC. In the in vivo study, the mice with candidiasis were treated successfully by a combination therapy of HNK with FLC, the results showed a decrease of the colony forming unit in infected and treated animals compared to the controls, at the conditions of the treatment used in this study. Synergistic activity of HNK and FLC against clinical isolates of FLC-resistant C. albicans was observed in vitro and in vivo. This report might provide a potential therapeutic method to overcome the problem of drug-resistance in C. albicans.

Porcine sclera as a model of human sclera for in vitro transport experiments: histology, SEM, and comparative permeability

PubMed Central

Ferrari, G.; Quarta, M.; Macaluso, C.; Govoni, P.; Dallatana, D.; Santi, P.

2009-01-01

Purpose To evaluate porcine sclera as a model of human sclera for in vitro studies of transscleral drug delivery of both low and high molecular weight compounds. Methods Human and porcine scleras were characterized for thickness and water content. The tissue surface was examined by scanning electron microscopy (SEM), and the histology was studied with hematoxylin-eosin staining. Comparative permeation experiments were performed using three model molecules, acetaminophen as the model compound for small molecules; a linear dextran with a molecular weight of 120 kDa as the model compound for high molecular weight drugs; and insulin, which was chosen as the model protein. Permeation parameters such as flux, lag time, and permeability coefficient were determined and compared. Results Human and porcine scleras have a similar histology and collagen bundle organization. The water content is approx 70% for both tissues while a statistically significant difference was found for the thickness, porcine sclera being approximately twofold thicker than human sclera. Differences in thickness produced differences in the permeability coefficient. In fact, human sclera was found to be two to threefold more permeable toward the three molecules studied than porcine sclera. Conclusions The results obtained in the present paper prove that porcine sclera can be considered a good model for human sclera for in vitro permeation experiments of both low and high molecular weight compounds. In fact, if the different tissue thickness is taken into account, comparable permeability was demonstrated. This suggests a possible use of this model in the evaluation of the transscleral permeation of new biotech compounds, which currently represent the most innovative and efficient therapeutic options for the treatment of ocular diseases. PMID:19190734

Integrated Model of Chemical Perturbations of a Biological Pathway Using 18 In Vitro High-Throughput Screening Assays for the Estrogen Receptor.

PubMed

Judson, Richard S; Magpantay, Felicia Maria; Chickarmane, Vijay; Haskell, Cymra; Tania, Nessy; Taylor, Jean; Xia, Menghang; Huang, Ruili; Rotroff, Daniel M; Filer, Dayne L; Houck, Keith A; Martin, Matthew T; Sipes, Nisha; Richard, Ann M; Mansouri, Kamel; Setzer, R Woodrow; Knudsen, Thomas B; Crofton, Kevin M; Thomas, Russell S

2015-11-01

We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation, and ER-dependent cell proliferation. The network model uses activity patterns across the in vitro assays to predict whether a chemical is an ER agonist or antagonist, or is otherwise influencing the assays through a manner dependent on the physics and chemistry of the technology platform ("assay interference"). The method is applied to a library of 1812 commercial and environmental chemicals, including 45 ER positive and negative reference chemicals. Among the reference chemicals, the network model correctly identified the agonists and antagonists with the exception of very weak compounds whose activity was outside the concentration range tested. The model agonist score also correlated with the expected potency class of the active reference chemicals. Of the 1812 chemicals evaluated, 111 (6.1%) were predicted to be strongly ER active in agonist or antagonist mode. This dataset and model were also used to begin a systematic investigation of assay interference. The most prominent cause of false-positive activity (activity in an assay that is likely not due to interaction of the chemical with ER) is cytotoxicity. The model provides the ability to prioritize a large set of important environmental chemicals with human exposure potential for additional in vivo endocrine testing. Finally, this model is generalizable to any molecular pathway for which there are multiple upstream and downstream assays available. Published by Oxford University Press on behalf of the Society of Toxicology 2015. This work is written by US Government employees and is in the public domain in the US.

In vitro to In vivo extrapolation of hepatic metabolism in fish: An inter-laboratory comparison of In vitro methods

EPA Science Inventory

Chemical biotransformation represents the single largest source of uncertainty in chemical bioaccumulation assessments for fish. In vitro methods employing isolated hepatocytes and liver subcellular fractions (S9) can be used to estimate whole-body rates of chemical metabolism, ...

In vitro reconstruction of human junctional and sulcular epithelium

PubMed Central

Dabija-Wolter, G; Bakken, V; Cimpan, M R; Johannessen, A C; Costea, D E

2013-01-01

BACKGROUND The aim of this study was to develop and characterize standardized in vitro three-dimensional organotypic models of human junctional epithelium (JE) and sulcular epithelium (SE). METHODS Organotypic models were constructed by growing human normal gingival keratinocytes on top of collagen matrices populated with gingival fibroblasts (GF) or periodontal ligament fibroblasts (PLF). Tissues obtained were harvested at different time points and assessed for epithelial morphology, proliferation (Ki67), expression of JE-specific markers (ODAM and FDC-SP), cytokeratins (CK), transglutaminase, filaggrin, and basement membrane proteins (collagen IV and laminin1). RESULTS The epithelial component in 3- and 5-day organotypics showed limited differentiation and expressed Ki-67, ODAM, FDC-SP, CK 8, 13, 16, 19, and transglutaminase in a similar fashion to control JE samples. PLF supported better than GF expression of CK19 and suprabasal proliferation, although statistically significant only at day 5. Basement membrane proteins started to be deposited only from day 5. The rate of proliferating cells as well as the percentage of CK19-expressing cells decreased significantly in 7- and 9-day cultures. Day 7 organotypics presented higher number of epithelial cell layers, proliferating cells in suprabasal layers, and CK expression pattern similar to SE. CONCLUSION Both time in culture and fibroblast type had impact on epithelial phenotype. Five-day cultures with PLF are suggested as JE models, 7-day cultures with PLF or GF as SE models, while 9-day cultures with GF as gingival epithelium (GE) models. Such standard, reproducible models represent useful tools to study periodontal bacteria–host interactions in vitro. PMID:22947066

Cancer-associated fibroblast promote transmigration through endothelial brain cells in three-dimensional in vitro models.

PubMed

Choi, Yoon Pyo; Lee, Joo Hyun; Gao, Ming-Qing; Kim, Baek Gil; Kang, Suki; Kim, Se Hoon; Cho, Nam Hoon

2014-11-01

Brain metastases are associated with high morbidity as well as with poor prognosis and survival in breast cancer patients. Despite its clinical importance, metastasis of breast cancer cells through the blood-brain barrier (BBB) is poorly understood. The objective of our study was to investigate whether cancer-associated fibroblasts (CAFs) play crucial roles in breast cancer brain metastasis. Using a cell adhesion assays, in vitro BBB permeability and transmigration assays and soft agar colony formation assays, we investigated the physical roles of CAFs in breast cancer brain metastasis. We also performed immunofluorescence, flow cytometric analysis, Droplet Digital PCR and Simon™ Simple Western System to confirm changes in expression levels. We established two novel three-dimensional (3D) culture systems using a perpendicular slide chamber and applying 3D embedded culture method to reflect brain metastasis conditions. With a newly developed device, CAFs was proven to promote cell adhesion to human brain microvascular endothelial cells, in vitro BBB permeability and transmigration and colony formation of breast cancer cells. Furthermore, CAFs enhanced the invasive migration of breast cancer cells in two kinds of 3D cultures. These 3D models also reliably recapitulate the initial steps of BBB transmigration, micro-metastasis and colonization. Expression of integrin α5β1 and αvβ3, c-MET and α2,6-siayltransferase was increased in breast cancer cells that migrated through the BBB. In conclusion, based on our in vitro BBB and co-culture models, our data suggest that CAFs may play a role in breast cancer brain metastasis. © 2014 UICC.

A novel antiangiogenic peptide derived from hepatocyte growth factor inhibits neovascularization in vitro and in vivo

PubMed Central

Xu, Yi; Zhao, Hui; Zheng, Ying; Gu, Qing; Ma, Jianxing

2010-01-01

Purpose To study the antiangiogenic activity of two small peptides (H-RN and H-FT) derived from the hepatocyte growth factor kringle 1 domain (HGF K1) using in vitro and in vivo assays. Methods RF/6A rhesus macaque choroid-retina endothelial cells were used for in vitro studies. The inhibiting effect of two peptides on a vascular endothelial growth factor (VEGF)-stimulated cell proliferation, cell migration, and endothelial cell tube formation were investigated. For in vivo assays, the antiangiogenic activity of H-RN and H-FT in the chick chorioallantoic membrane model (CAM) and a mice oxygen-induced retinopathy model (OIR) were studied. A recombinant mouse VEGF-neutralizing antibody, bevacizumab, and a scrambled peptide were used as two control groups in separate studies. Results H-RN effectively inhibited VEGF-stimulated RF/6A cell proliferation, migration, and tube formation on Matrigel™, while H-FT did not. H-RN was also able to inhibit angiogenesis when applied to the CAM, and had antineovascularization activity in the retinal neovascularization of a mouse OIR model when administrated as an intravitreous injection. The antiangiogenic activity of H-RN was not as strong as that of VEGF antibodies. The H-FT and scrambled peptide had no such activity. Conclusions H-RN, a new peptide derived from the HGF K1 domain, was shown to have antiangiogenic activity in vitro and in vivo. It may lead to new potential drug discoveries and the development of new treatments for pathological retinal angiogenesis. PMID:21031024

In vitro and in vivo radiosensitization induced by hydroxyapatite nanoparticles

PubMed Central

Chu, Sheng-Hua; Karri, Surya; Ma, Yan-Bin; Feng, Dong-Fu; Li, Zhi-Qiang

2013-01-01

Background Previous study showed that hydroxyapatite nanoparticles (nano-HAPs) inhibited glioma growth in vitro and in vivo; and in a drug combination, they could reduce adverse reactions. We investigated the possible enhancement of radiosensitivity induced by nano-HAPs. Methods In vitro radiosensitization of nano-HAPs was measured using a clonogenic survival assay in human glioblastoma U251 and breast tumor brain metastatic tumor MDA-MB-231BR cells. DNA damage and repair were measured using γH2AX foci, and mitotic catastrophe was determined by immunostaining. The effect of nano-HAPs on in vivo tumor radiosensitivity was investigated in a subcutaneous and an orthotopic model. Results Nano-HAPs enhanced each cell line's radiosensitivity when the exposure was 1 h before irradiation, and they had no significant effect on irradiation-induced apoptosis or on the activation of the G2 cell cycle checkpoint. The number of γH2AX foci per cell was significantly large at 24 h after the combination modality of nano-HAPs + irradiation compared with single treatments. Mitotic catastrophe was also significantly increased at an interval of 72 h in tumor cells receiving the combined modality compared with the individual treatments. In a subcutaneous model, nano-HAPs caused a larger than additive increase in tumor growth delay. In an orthotopic model, nano-HAPs significantly reduced tumor growth and extended the prolongation of survival induced by irradiation. Conclusions These results show that nano-HAPs can enhance the radiosensitivity of tumor cells in vitro and in vivo through the inhibition of DNA repair, resulting in an increase in mitotic catastrophe. PMID:23519742

Hepatoprotective and antioxidant activity of quercetin loaded chitosan/alginate particles in vitro and in vivo in a model of paracetamol-induced toxicity.

PubMed

Tzankova, Virginia; Aluani, Denitsa; Kondeva-Burdina, Magdalena; Yordanov, Yordan; Odzhakov, Feodor; Apostolov, Alexandar; Yoncheva, Krassimira

2017-08-01

The toxic liver impairment caused by free radical injury or excessive reactive oxigen species (ROS) formation could be effectivelly attenuated by natural antioxidants. The present study aimed to explore and compare the hepatoprotective and antioxidant effects of free and encapsulated quercetin in in vitro and in vivo models of hepatotoxicity. Thus, quercetin was encapsulated in chitosan/alginate nanoparticles by gelation method. Both empty and quercetin-loaded nanoparticles revealed good safety profile in vitro, determined by the lack of cytotoxicity in human hepatoma HepG2 cells. The pretreatment of HepG2 cells with encapsulated quercetin (10μg/ml) significantly attenuated the decrease in cell viability in H 2 О 2 -induced oxidative stress (0.1mM H 2 О 2 ) , thus showing an effective in vitro protection. In vivo evaluation of the antioxidant and hepatoprotective potential of free and encapsulated quercetin was performed in a model of paracetamol - induced liver injury in male Wistar rats. The oral pretreatment with encapsulated quercetin (0.18mg/kg b.w., 7days) significantly diminished the increased levels of serum transaminases ALT and AST, attenuated the lipid peroxidation and restored the levels of gluthation (a marker of cell antioxidant defence system). The protective effects of quercetin encapsulated in chitosan-based nanoformulation were superior to those of free quercetin. The results of the study suggest that the encapsulation of quercetin in chitosan/alginate nanoformulations might represent an effective therapeutic approach against oxidative stress induced liver injury. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Targeting regenerative exosomes to myocardial infarction using cardiac homing peptide

PubMed Central

Vandergriff, Adam; Huang, Ke; Shen, Deliang; Hu, Shiqi; Hensley, Michael Taylor; Caranasos, Thomas G.; Qian, Li; Cheng, Ke

2018-01-01

Rationale: Cardiac stem cell-derived exosomes have been demonstrated to promote cardiac regeneration following myocardial infarction in preclinical studies. Recent studies have used intramyocardial injection in order to concentrate exosomes in the infarct. Though effective in a research setting, this method is not clinically appealing due to its invasive nature. We propose the use of a targeting peptide, cardiac homing peptide (CHP), to target intravenously-infused exosomes to the infarcted heart. Methods: Exosomes were conjugated with CHP through a DOPE-NHS linker. Ex vivo targeting was analyzed by incubating organ sections with the CHP exosomes and analyzing with fluorescence microscopy. In vitro assays were performed on neonatal rat cardiomyocytes and H9C2 cells. For the animal study, we utilized an ischemia/reperfusion rat model. Animals were treated with either saline, scramble peptide exosomes, or CHP exosomes 24 h after surgery. Echocardiography was performed 4 h after surgery and 21 d after surgery. At 21 d, animals were sacrificed, and organs were collected for analysis. Results: By conjugating the exosomes with CHP, we demonstrate increased retention of the exosomes within heart sections ex vivo and in vitro with neonatal rat cardiomyocytes. In vitro studies showed improved viability, reduced apoptosis and increased exosome uptake when using CHP-XOs. Using an animal model of ischemia/reperfusion injury, we measured the heart function, infarct size, cellular proliferation, and angiogenesis, with improved outcomes with the CHP exosomes. Conclusions: Our results demonstrate a novel method for increasing delivery of for treatment of myocardial infarction. By targeting exosomes to the infarcted heart, there was a significant improvement in outcomes with reduced fibrosis and scar size, and increased cellular proliferation and angiogenesis. PMID:29556361

Evaluation of antioxidant potential of essential oils of some commonly used Indian spices in in vitro models and in food supplements enriched with omega-6 and omega-3 fatty acids.

PubMed

Bag, Anwesa; Chattopadhyay, Rabi Ranjan

2018-01-01

The aim of this study was to evaluate and compare the antioxidant potential of essential oils of some commonly used Indian spices (black pepper, cinnamon, clove, coriander and cumin) in various in vitro models and in food supplements enriched with omega-6 and omega-3 fatty acids. In vitro antioxidant potential was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging and Fe 2+ ion-chelating methods and lipid oxidation stabilisation potential was evaluated in bulk soybean oil-fish oil mixture and their oil-in-water emulsions using peroxide value (PV), p-anisidine value (p-AV) and total oxidation value as indicators of oxidation. Combination effects using DPPH radical scavenging and Briggs-Rauscher oscillating reaction methods were also evaluated. Test essential oils showed varying degrees of radical scavenging and Fe 2+ ion-chelating efficacy. Clove and coriander oils showed significantly higher (P < 0.05) radical scavenging and Fe 2+ ion-chelating potential over other tested essential oils as well as BHT and ∞-tocopherol. The anti-lipid peroxidative potential of test essential oils was found in the following decreasing order: clove > coriander > BHT > cinnamon > α-tocopherol > cumin > black pepper. Furthermore, clove and coriander oils showed synergistic antioxidant activity in combination both in DPPH radical scavenging and Briggs-Rauscher oscillating reaction methods whereas other possible combinations showed additive effects. Strong radical scavenging and Fe 2+ -chelating as well as anti-lipid peroxidative activities of clove and coriander oils provide evidence that clove and coriander oils may serve as a potential source of natural antioxidants for retarding lipid oxidation of food supplements enriched with omega-6 and omega-3 fatty acids.

Reconstitution of mouse oogenesis in a dish from pluripotent stem cells.

PubMed

Hayashi, Katsuhiko; Hikabe, Orie; Obata, Yayoi; Hirao, Yuji

2017-09-01

This protocol is an extension to: Nat. Protoc. 8, 1513-1524 (2013); doi: 10.1038/nprot.2013.090; published online 11 July 2013Generation of functional oocytes in culture from pluripotent stem cells should provide a useful model system for improving our understanding of the basic mechanisms underlying oogenesis. In addition, it has potential applications as an alternative source of oocytes for reproduction. Using the most advanced mouse model in regard to reproductive engineering and stem cell biology, we previously developed a culture method that produces functional primorial germ cells starting from pluripotent cells in culture and described it in a previous protocol. This Protocol Extension describes an adaptation of this existing Protocol in which oogenesis also occurs in vitro, thus substantially modifying the technique. Oocytes generated from embryonic stem cells (ESCs) or induced pluripotent stem cells give rise to healthy pups. Here, we describe the protocol for oocyte generation in culture. The protocol is mainly composed of three different culture stages: in vitro differentiation (IVDi), in vitro growth (IVG), and in vitro maturation (IVM), which in total take ∼5 weeks. In each culture period, there are several checkpoints that enable the number of oocytes being produced in the culture to be monitored. The basic structure of the culture system should provide a useful tool for clarifying the complicated sequence of oogenesis in mammals.

Coupled Analysis of In Vitro and Histology Tissue Samples to Quantify Structure-Function Relationship

PubMed Central

Acar, Evrim; Plopper, George E.; Yener, Bülent

2012-01-01

The structure/function relationship is fundamental to our understanding of biological systems at all levels, and drives most, if not all, techniques for detecting, diagnosing, and treating disease. However, at the tissue level of biological complexity we encounter a gap in the structure/function relationship: having accumulated an extraordinary amount of detailed information about biological tissues at the cellular and subcellular level, we cannot assemble it in a way that explains the correspondingly complex biological functions these structures perform. To help close this information gap we define here several quantitative temperospatial features that link tissue structure to its corresponding biological function. Both histological images of human tissue samples and fluorescence images of three-dimensional cultures of human cells are used to compare the accuracy of in vitro culture models with their corresponding human tissues. To the best of our knowledge, there is no prior work on a quantitative comparison of histology and in vitro samples. Features are calculated from graph theoretical representations of tissue structures and the data are analyzed in the form of matrices and higher-order tensors using matrix and tensor factorization methods, with a goal of differentiating between cancerous and healthy states of brain, breast, and bone tissues. We also show that our techniques can differentiate between the structural organization of native tissues and their corresponding in vitro engineered cell culture models. PMID:22479315

Effect of sperm entry on blastocyst development after in vitro fertilization and intracytoplasmic sperm injection - mouse model.

PubMed

Piotrowska-Nitsche, Karolina; Chan, Anthony W S

2013-01-01

To investigate whether in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), influence the embryo's development and its quality using the mouse as a model. Assisted fertilization was performed using ICSI and IVF. Fluorescent beads were adhered to the fertilization cone or place of previous sperm injection in the natural mated (NM), IVF and ICSI embryos, respectively. Embryo examination was carried out at the two-cell and blastocyst stage to determine the position of fluorescent bead. Protein expression was detected by fluorescence immunocytochemical staining and confocal microscopic imaging of blastocysts. IVF and ICSI embryos developed at rates comparable to NM group. Embryos show similar expression patterns of two transcription factors, Oct4 and Cdx2. The most preferred place for spermatozoa attachment was the equatorial site of the egg, whether fertilization occurred in vitro or under natural conditions. We also link the sperm entry position (SEP) to embryo morphology and the number of cells at the blastocyst stage, with no influence of the method of fertilization. IVF and ICSI, do not compromise in vitro pre-implantation development. Additional data, related to sperm entry, could offer further criteria to predict embryos that will implant successfully. Based on embryo morphology, developmental rate and protein expression level of key transcription factors, our results support the view that ART techniques, such as IVF and ICSI, do not perturb embryonic development or quality.

Establishment of an in vitro photoassay using THP-1 cells and IL-8 to discriminate photoirritants from photoallergens.

PubMed

Martínez, V; Galbiati, V; Corsini, E; Martín-Venegas, R; Vinardell, M P; Mitjans, M

2013-09-01

At present, there are no in vivo or in vitro methods developed which has been adopted by regulatory authorities to assess photosensitization induced by chemicals. Recently, we have proposed the use of THP-1 cells and IL-8 release to identify the potential of chemicals to induce skin sensitization. Based on the assumption that sensitization and photosensitization share common mechanisms, the aim of this work was to explore the THP-1 model as an in vitro model to identify photoallergenic chemicals. THP-1 cells were exposed to 7 photoallergens and 3 photoirritants and irradiated with UVA light or kept in dark. Non phototoxic allergens or irritants were also included as negative compounds. Following 24h of incubation, cytotoxicity and IL-8 release were measured. At subtoxic concentrations, photoallergens produced a dose-related increase in IL-8 release after irradiation. Some photoirritants also produced a slight increase in IL-8 release. However, when the overall stimulation indexes of IL-8 were calculated for each chemical, 6 out of 7 photoallergens tested reached a stimulation index above 2, while the entire set of negative compounds had stimulation indexes below 2. Our data suggest that this assay may become a useful cell-based in vitro test for evaluating the photosensitizing potential of chemicals. Copyright © 2013 Elsevier Ltd. All rights reserved.

Generation of multicellular tumor spheroids by the hanging-drop method.

PubMed

Timmins, Nicholas E; Nielsen, Lars K

2007-01-01

Owing to their in vivo-like characteristics, three-dimensional (3D) multicellular tumor spheroid (MCTS) cultures are gaining increasing popularity as an in vitro model of tumors. A straightforward and simple approach to the cultivation of these MCTS is the hanging-drop method. Cells are suspended in droplets of medium, where they develop into coherent 3D aggregates and are readily accessed for analysis. In addition to being simple, the method eliminates surface interactions with an underlying substratum (e.g., polystyrene plastic or agarose), requires only a low number of starting cells, and is highly reproducible. This method has also been applied to the co-cultivation of mixed cell populations, including the co-cultivation of endothelial cells and tumor cells as a model of early tumor angiogenesis.

Modification of lymphocyte responsiveness in vitro by lambda carrageenan compared with colloidal silica and depletion of surface adherent cells.

PubMed Central

Pawelec, G; Brons, G

1978-01-01

The effects of the immunosuppressive sulphated polygalactan lambda carrageenan on in vitro models of allograft immunity were compared with the effects of removing macrophages (surface adherent and/or phagocytic cells) by established methods. Carrageenan depressed the primary mixed lymphocyte reactions, but not to the same extent as the removal of macrophages. 2-Mercaptoethanol restored the response. Secondary mixed lymphocyte reactions and responses to phytohaemaglutinin were depressed by carrageenan but not by the removal of macrophages, and in these systems 2-mercaptoethanol failed to restore the responses of carrageenan-treated cultures. In contrast, cell-mediated cytolysis by presensitized lymphocytes was not affected by carrageenan or by colloidal silica. Carrageenan depressed cell-mediated cytolysis only if it was present during the sensitization of the effector cells. We conclude that carrageenan can have two dose-related effects in vitro: one on the macrophage and one on the responding lymphocyte. PMID:207475

Bioreactor Cultivation of Anatomically Shaped Human Bone Grafts

PubMed Central

Temple, Joshua P.; Yeager, Keith; Bhumiratana, Sarindr; Vunjak-Novakovic, Gordana; Grayson, Warren L.

2015-01-01

In this chapter, we describe a method for engineering bone grafts in vitro with the specific geometry of the temporomandibular joint (TMJ) condyle. The anatomical geometry of the bone grafts was segmented from computed tomography (CT) scans, converted to G-code, and used to machine decellularized trabecular bone scaffolds into the identical shape of the condyle. These scaffolds were seeded with human bone marrow-derived mesenchymal stem cells (MSCs) using spinner flasks and cultivated for up to 5 weeks in vitro using a custom-designed perfusion bioreactor system. The flow patterns through the complex geometry were modeled using the FloWorks module of SolidWorks to optimize bioreactor design. The perfused scaffolds exhibited significantly higher cellular content, better matrix production, and increased bone mineral deposition relative to non-perfused (static) controls after 5 weeks of in vitro cultivation. This technology is broadly applicable for creating patient-specific bone grafts of varying shapes and sizes. PMID:24014312

Formation of gut-like structures in vitro from mouse embryonic stem cells.

PubMed

Torihashi, Shigeko

2006-01-01

Embryonic stem (ES) cells have the potential to differentiate into all cell types originating from the three germ layers; however, there are still few reports about the formation of functional organs from embryonic stem cells. Recently, we reported that by hanging drops of mouse ES cells, embryoid bodies (EBs) formed gut-like structures in vitro composed of three layers corresponding to the epithelium, lamina propria, and musculature. The morphological features and the process of formation are similar to gut and its organogenesis in vivo. Thus, this is a good model for development of the gut and a useful tool for analysis of the factors required for gut organogenesis. The protocol basically involves a method of hanging drops to make EBs, which are then plated on coated dishes for outgrowth. EBs develop to form gut-like structures when induced to spontaneously enter a program of differentiation in vitro without addition of any extrinsic factors.

Generation of Cardiomyocytes from Pluripotent Stem Cells.

PubMed

Nakahama, Hiroko; Di Pasquale, Elisa

2016-01-01

The advent of pluripotent stem cells (PSCs) enabled a multitude of studies for modeling the development of diseases and testing pharmaceutical therapeutic potential in vitro. These PSCs have been differentiated to multiple cell types to demonstrate its pluripotent potential, including cardiomyocytes (CMs). However, the efficiency and efficacy of differentiation vary greatly between different cell lines and methods. Here, we describe two different methods for acquiring CMs from human pluripotent lines. One method involves the generation of embryoid bodies, which emulates the natural developmental process, while the other method chemically activates the canonical Wnt signaling pathway to induce a monolayer of cardiac differentiation.

Quantifying Spiral Ganglion Neurite and Schwann Behavior on Micropatterned Polymer Substrates.

PubMed

Cheng, Elise L; Leigh, Braden; Guymon, C Allan; Hansen, Marlan R

2016-01-01

The first successful in vitro experiments on the cochlea were conducted in 1928 by Honor Fell (Fell, Arch Exp Zellforsch 7(1):69-81, 1928). Since then, techniques for culture of this tissue have been refined, and dissociated primary culture of the spiral ganglion has become a widely accepted in vitro model for studying nerve damage and regeneration in the cochlea. Additionally, patterned substrates have been developed that facilitate and direct neural outgrowth. A number of automated and semi-automated methods for quantifying this neurite outgrowth have been utilized in recent years (Zhang et al., J Neurosci Methods 160(1):149-162, 2007; Tapias et al., Neurobiol Dis 54:158-168, 2013). Here, we describe a method to study the effect of topographical cues on spiral ganglion neurite and Schwann cell alignment. We discuss our microfabrication process, characterization of pattern features, cell culture techniques for both spiral ganglion neurons and spiral ganglion Schwann cells. In addition, we describe protocols for reducing fibroblast count, immunocytochemistry, and methods for quantifying neurite and Schwann cell alignment.

Ultrasound SIV measurement of helical valvular flow behind the great saphenous vein

NASA Astrophysics Data System (ADS)

Park, Jun Hong; Kim, Jeong Ju; Lee, Sang Joon; Yeom, Eunseop; Experimental Fluid Mechanics Laboratory Team; LaboratoryMicrothermal; Microfluidic Measurements Collaboration

2017-11-01

Dysfunction of venous valve and induced secondary abnormal flow are closely associated with venous diseases. Thus, detailed analysis of venous valvular flow is invaluable from biological and medical perspectives. However, most previous studies on venous perivalvular flows were based on qualitative analyses. On the contrary, quantitative analysis on the perivalvular flows has not been fully understood yet. In this study, 3D valvular flows under in vitro and in vivo conditions were experimentally investigated using ultrasound speckle image velocimetry (SIV) for analyzing their flow characteristics. The results for in vitro model obtained by the SIV technique were compared with those derived by numerical simulation and color Doppler method to validate its measurement accuracy. Then blood flow in the human great saphenous vein was measured using the SIV with respect to the dimensionless index, helical intensity. The results obtained by the SIV method are well matched well with those obtained by the numerical simulation and color Doppler method. The hemodynamic characteristics of 3D valvular flows measured by the validated SIV method would be helpful in diagnosis of valve-related venous diseases. None.

Differentiation and characterization of human pluripotent stem cell-derived brain microvascular endothelial cells.

PubMed

Stebbins, Matthew J; Wilson, Hannah K; Canfield, Scott G; Qian, Tongcheng; Palecek, Sean P; Shusta, Eric V

2016-05-15

The blood-brain barrier (BBB) is a critical component of the central nervous system (CNS) that regulates the flux of material between the blood and the brain. Because of its barrier properties, the BBB creates a bottleneck to CNS drug delivery. Human in vitro BBB models offer a potential tool to screen pharmaceutical libraries for CNS penetration as well as for BBB modulators in development and disease, yet primary and immortalized models respectively lack scalability and robust phenotypes. Recently, in vitro BBB models derived from human pluripotent stem cells (hPSCs) have helped overcome these challenges by providing a scalable and renewable source of human brain microvascular endothelial cells (BMECs). We have demonstrated that hPSC-derived BMECs exhibit robust structural and functional characteristics reminiscent of the in vivo BBB. Here, we provide a detailed description of the methods required to differentiate and functionally characterize hPSC-derived BMECs to facilitate their widespread use in downstream applications. Copyright © 2015 Elsevier Inc. All rights reserved.

In vitro dynamic model simulating the digestive tract of 6-month-old infants

PubMed Central

Gallo, Marianna; Tornatore, Fabio; Frasso, Annalisa; Saccone, Giulia; Budelli, Andrea; Barone, Maria V.

2017-01-01

Background In vivo assays cannot always be conducted because of ethical reasons, technical constraints or costs, but a better understanding of the digestive process, especially in infants, could be of great help in preventing food-related pathologies and in developing new formulas with health benefits. In this context, in vitro dynamic systems to simulate human digestion and, in particular, infant digestion could become increasingly valuable. Objective To simulate the digestive process through the use of a dynamic model of the infant gastroenteric apparatus to study the digestibility of starch-based infant foods. Design Using M.I.D.A (Model of an Infant Digestive Apparatus), the oral, gastric and intestinal digestibility of two starch-based products were measured: 1) rice starch mixed with distilled water and treated using two different sterilization methods (the classical method with a holding temperature of 121°C for 37 min and the HTST method with a holding temperature of 137°C for 70 sec) and 2) a rice cream with (premium product) or without (basic product) an aliquot of rice flour fermented by Lactobacillus paracasei CBA L74. After the digestion the foods were analyzed for the starch concentration, the amount of D-glucose released and the percentage of hydrolyzed starch. Results An in vitro dynamic system, which was referred to as M.I.D.A., was obtained. Using this system, the starch digestion occurred only during the oral and intestinal phase, as expected. The D-glucose released during the intestinal phase was different between the classical and HTST methods (0.795 grams for the HTST versus 0.512 for the classical product). The same analysis was performed for the basic and premium products. In this case, the premium product had a significant difference in terms of the starch hydrolysis percentage during the entire process. Conclusions The M.I.D.A. system was able to digest simple starches and a more complex food in the correct compartments. In this study, better digestibility of the premium product was revealed. PMID:29261742

Development of a modified in vitro skin absorption method to study the epidermal/dermal disposition of a contact allergen in human skin.

PubMed

Pendlington, Ruth U; Minter, Helen J; Stupart, Leanne; MacKay, Cameron; Roper, Clive S; Sanders, David J; Pease, Camilla K

2008-01-01

In vitro skin absorption methods exist in Organisation for Economic Co-operation and Development (OECD) guideline form (No. 428) and are used to estimate the degree of systemic penetration of chemicals through skin. More detailed kinetics of permeation through skin compartments are not described well by existing methods. This study was designed to assess the practical feasibility of generating compartmental (stratum corneum/epidermal/dermal) disposition and kinetic data of topically applied chemicals. For chemically induced effects initiated in the skin (e.g., skin allergy), the delivery of tissue concentrations of chemical will impact the incidence and severity of biological effect. Explicit data on the kinetics of chemical disposition in skin have not traditionally been needed for skin allergy risk assessment: current in vivo assays embody delivery implicitly. Under the 7th Amendment to the European Cosmetics Directive, in vivo assays (such as the local lymph node assay for skin sensitization) will not be permitted to assess cosmetic ingredients. New in vitro and in silico alternative approaches and ways of predicting risk of adverse effects in humans need to be developed, and new methods such as that described here provide a way of estimating delivered concentrations and the effect of formulation changes on that delivery. As we continue to deconstruct the contributing factors of skin allergy in humans, it will be useful to have methods available that can measure skin tissue compartment exposure levels delivered from different exposure use scenarios. Here we provide such a method. The method could also be used to generate useful data for developing in silico kinetic models of compartmental skin delivery and for refining data for skin delivery in relation to the evaluation of systemic toxicity.

Study on the release of fenofibrate nanosuspension in vitro and its correlation with in situ intestinal and in vivo absorption kinetics in rats.

PubMed

Xu, Yuanlong; Wang, Yonglu; Li, Xue Ming; Huang, Qinqin; Chen, Wei; Liu, Ran; Chen, BaoAn; Wei, Ping

2014-07-01

As an oral delivery carrier for poorly water soluble drugs, the nanosuspension was prepared by melt emulsification method combined with high-pressure homogenization. The objective of this study was to clarify the absorption mechanism in rats of fenofibrate nanosuspension using the model of in situ gut perfusion. The release rate of drug from nanosuspension was fast which about 70% of the drug would be released within 5 minutes. The absorption of fenofibrate nanosuspension in the gastrointestinal (GI) tract was studied by the in situ closed loop method in rats. It was found that the absorption process in intestine was first-process with passive diffusion mechanism, and the whole intestine was the major segment for the drug absorption. Additionally, GI absorption in situ studies indicated that the fenofibrate nanosuspension had great success in regard to enhancement of intestinal absorption compared to the fenofibrate suspension of coarse powder. The pharmacokinetic characteristics were studied in rats after oral administration of fenofibrate nanosuspension or suspension at the dosage of 27 mg/kg. The plasma concentration-time curve was fitted to the one-compartment model. The correlation between in vitro dissolution (P), in situ intestinal absorption (F) and in vivo absorption (Fa) in rats was investigated with the results as follows: Fa = 6.2061P-456.38(r = 0.9559), F = 3.6911P-2.2169(r = 0.970), F = 0.5095P + 44.189(r = 0.9609). The highest level A could be obtained from the in vitro--in vivo correlation (IVIVC) between dissolution percentage and intestinal absorption of the fenofibrate nanosuspension in rats. Consequently, the in situ intestinal perfusion model could be used to predict the in vivo pharmacokinetic characteristics in rats.

In vitro cleaning potential of three different implant debridement methods.

PubMed

Sahrmann, Philipp; Ronay, Valerie; Hofer, Deborah; Attin, Thomas; Jung, Ronald E; Schmidlin, Patrick R

2015-03-01

To assess the cleaning potential of three different instrumentation methods commonly used for implant surface decontamination in vitro, using a bone defect-simulating model. Dental implants were stained with indelible ink and mounted in resin models, which represented standardized peri-implantitis defects with different bone defect angulations (30, 60 and 90°). Cleaning procedures were performed by either an experienced dental hygienist or a 2nd-year postgraduate student. The treatment was repeated 20 times for each instrumentation, that is, with a Gracey curette, an ultrasonic device and an air powder abrasive device (PAD) with glycine powder. After each run, implants were removed and images were taken to detect color remnants in order to measure planimetrically the cumulative uncleaned surface area. SEM images were taken to assess micromorphologic surface changes (magnification 10,000 ×). Results were tested for statistical differences using two-way ANOVA and Bonferroni correction. The areas of uncleaned surfaces (%, mean ± standard deviations) for curettes, ultrasonic tips, and airflow accounted for 24.1 ± 4.8%, 18.5 ± 3.8%, and 11.3 ± 5.4%, respectively. These results were statistically significantly different (P < 0.0001). The cleaning potential of the airflow device increased with wider defects. SEM evaluation displayed distinct surface alterations after instrumentation with steel tips, whereas glycine powder instrumentation had only a minute effect on the surface topography. Within the limitations of the present in vitro model, airflow devices using glycine powders seem to constitute an efficient therapeutic option for the debridement of implants in peri-implantitis defects. Still, some uncleaned areas remained. In wide defects, differences between instruments are more accentuated. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

In Vitro Model for Predicting the Protective Effect of Ultraviolet-Blocking Contact Lens in Human Corneal Epithelial Cells.

PubMed

Abengózar-Vela, Antonio; Arroyo, Cristina; Reinoso, Roberto; Enríquez-de-Salamanca, Amalia; Corell, Alfredo; González-García, María Jesús

2015-01-01

To develop an in vitro method to determine the protective effect of UV-blocking contact lenses (CLs) in human corneal epithelial (HCE) cells exposed to UV-B radiation. SV-40-transformed HCE cells were covered with non-UV-blocking CL, UV-blocking CL or not covered, and exposed to UV-B radiation. As control, HCE cells were covered with both types of CLs or not covered, but not exposed to UV-B radiation. Cell viability at 24, 48 and 72 h, after UV-B exposure and removing CLs, was determined by alamarBlue(®) assay. Percentage of live, dead and apoptotic cells was also assessed by flow cytometry after 24 h of UV-B exposure. Intracellular reactive oxygen species (ROS) production after 1 h of exposure was assessed using the dye H(2)DCF-DA. Cell viability significantly decreased, apoptotic cells and intracellular ROS production significantly increased when UVB-exposed cells were covered with non-UV-blocking CL or not covered compared to non-irradiated cells. When cells were covered with UV-blocking CL, cell viability significantly increased and apoptotic cells and intracellular ROS production did not increase compared to exposed cells. UV-B radiation induces cell death by apoptosis, increases ROS production and decreases viable cells. UV-blocking CL is able to avoid these effects increasing cell viability and protecting HCE cells from apoptosis and ROS production induced by UV-B radiation. This in vitro model is an alternative to in vivo methods to determine the protective effect of UV-blocking ophthalmic biomaterials because it is a quicker, cheaper and reliable model that avoids the use of animals.

Cryopreservation and in vitro culture of primary cell types from lung tissue of a stranded pygmy sperm whale (Kogia breviceps).

PubMed

Annalaura Mancia; Spyropoulos, Demetri D; McFee, Wayne E; Newton, Danforth A; Baatz, John E

2012-01-01

Current models for in vitro studies of tissue function and physiology, including responses to hypoxia or environmental toxins, are limited and rely heavily on standard 2-dimensional (2-D) cultures with immortalized murine or human cell lines. To develop a new more powerful model system, we have pursued methods to establish and expand cultures of primary lung cell types and reconstituted tissues from marine mammals. What little is known about the physiology of the deep-sea diving pygmy sperm whale (PSW), Kogia breviceps, comes primarily from stranding events that occur along the coast of the southeastern United States. Thus, development of a method for preserving live tissues and retrieving live cells from deceased stranded individuals was initiated. This report documents successful cryopreservation of PSW lung tissue. We established in vitro cultures of primary lung cell types from tissue fragments that had been cryopreserved several months earlier at the stranding event. Dissociation of cryopreserved lung tissues readily provides a variety of primary cell types that, to varying degrees, can be expanded and further studied/manipulated in cell culture. In addition, PSW-specific molecular markers have been developed that permitted the monitoring of fibroblast, alveolar type II, and vascular endothelial cell types. Reconstitution of 3-D cultures of lung tissues with these cell types is now underway. This novel system may facilitate the development of rare or disease-specific lung tissue models (e.g., to test causes of PSW stranding events and lead to improved treatments for pulmonary hypertension or reperfusion injury in humans). Also, the establishment of a "living" tissue bank biorepository for rare/endangered species could serve multiple purposes as surrogates for freshly isolated samples. Copyright © 2011 Elsevier Inc. All rights reserved.

A first step towards a consensus static in vitro model for simulating full-term infant digestion.

PubMed

Ménard, O; Bourlieu, C; De Oliveira, S C; Dellarosa, N; Laghi, L; Carrière, F; Capozzi, F; Dupont, D; Deglaire, A

2018-02-01

In vitro alternatives to clinical trials are used for studying human food digestion. For simulating infant digestion, only a few models, lacking physiological relevance, are available. Thanks to an extensive literature review of the in vivo infant digestive conditions, a gastrointestinal static in vitro model was developed for infants born at term and aged 28days. The model was applied to the digestion of a commercial infant formula. Kinetics of digestion, as well as the structural evolution, were compared with those obtained while submitting the same formula to the adult international consensus protocol of in vitro static digestion. The kinetics of proteolysis and lipolysis differed according to the physiological stage resulting mainly from the reduced level of enzymes and bile salts, as well as the higher gastric pH in the infant model. This in vitro static model of infant digestion is of interest for scientists, food or pharmaceutical manufacturers. Copyright © 2017 Elsevier Ltd. All rights reserved.

In vitro pharmacokinetic/pharmacodynamic models in anti-infective drug development: focus on TB

PubMed Central

Vaddady, Pavan K; Lee, Richard E; Meibohm, Bernd

2011-01-01

For rapid anti-tuberculosis (TB) drug development in vitro pharmacokinetic/pharmacodynamic (PK/PD) models are useful in evaluating the direct interaction between the drug and the bacteria, thereby guiding the selection of candidate compounds and the optimization of their dosing regimens. Utilizing in vivo drug-clearance profiles from animal and/or human studies and simulating them in an in vitro PK/PD model allows the in-depth characterization of antibiotic activity of new and existing antibacterials by generating time–kill data. These data capture the dynamic interplay between mycobacterial growth and changing drug concentration as encountered during prolonged drug therapy. This review focuses on important PK/PD parameters relevant to anti-TB drug development, provides an overview of in vitro PK/PD models used to evaluate the efficacy of agents against mycobacteria and discusses the related mathematical modeling approaches of time–kill data. Overall, it provides an introduction to in vitro PK/PD models and their application as critical tools in evaluating anti-TB drugs. PMID:21359155

Coupling of computer modeling with in vitro methodologies to reduce animal usage in toxicity testing.

PubMed

Clewell, H J

1993-05-01

The use of in vitro data to support the development of physiologically based pharmacokinetic (PBPK) models and to reduce the requirement for in vivo testing is demonstrated by three examples. In the first example, polychlorotrifluoroethylene, in vitro studies comparing metabolism and tissue response in rodents and primates made it possible to obtain definitive data for a human risk assessment without resorting to additional in vivo studies with primates. In the second example, a PBPK model for organophosphate esters was developed in which the parameters defining metabolism, tissue partitioning, and enzyme inhibition were all characterized by in vitro studies, and the rest of the model parameters were established from the literature. The resulting model was able to provide a coherent description of enzyme inhibition following both acute and chronic exposures in mice, rats, and humans. In the final example, the carcinogenic risk assessment for methylene chloride was refined by the incorporation of in vitro data on human metabolism into a PBPK model.

Novel paclitaxel-coated angioplasty balloon catheter based on cetylpyridinium salicylate: preparation, characterization and simulated use in an in vitro vessel model.

PubMed

Petersen, Svea; Kaule, Sebastian; Stein, Florian; Minrath, Ingo; Schmitz, Klaus-Peter; Kragl, Udo; Sternberg, Katrin

2013-10-01

Drug-coated balloons (DCB), which have emerged as therapeutic alternative to drug-eluting stents in percutaneous cardiovascular intervention, are well described with regard to clinical efficiency and safety within a number of clinical studies. In vitro studies elucidating the correlation of coating method and composition with DCB performance are however rare but considered important for the understanding of DCB requirements and the improvement of established DCB. In this context, we evaluated the applicability of a pipetting, dip-coating, and spray-coating process for the establishment of DCB based on paclitaxel (PTX) and the ionic liquid cetylpyridinium salicylate (Cetpyrsal) as novel innovative additive in three different compositions. Among tested methods and compositions, the pipetting process with 50 wt.% PTX resulted in most promising coatings as drug load was less controllable by the other processes and higher PTX contents led to considerable drug crystallization, as visualized by electron microscopy, accelerating PTX loss during short-term elution. Applying these conditions, homogeneous coatings could be applied on balloon catheter, whose simulated use in an in vitro vessel model revealed percental drug losses of 36 and 28% during transit and percental drug transfers of 12 and 40% under expansion for coatings applied in expanded and folded balloon condition, respectively. In comparison to literature values, these results support the high potential of Cetpyrsal as novel DCB matrix regarding low drug loss and efficient drug transfer. © 2013.

Sun light mediated synthesis of gold nanoparticles as carrier for 6-mercaptopurine: Preparation, characterization and toxicity studies in zebrafish embryo model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganeshkumar, Moorthy; Sastry, Thotapalli Parvathaleswara; Sathish Kumar, Muniram

2012-09-15

Highlights: ► Gold nanoparticles prepared using eco-friendly method with good in vitro stability. ► Can be used as drug delivery system. ► Did not show any toxicity in zebrafish embryo. ► More toxic to cancer cells when compared to N-Au-Mp and Mp. -- Abstract: The objective of this study is to synthesize green chemistry based gold nanoparticles by sun light irradiation method. The prepared gold nanoparticles (AuNPs) were modified using folic acid and then coupled with 6-mercaptopurine. These modified nanoparticles were used as a tool for targeted drug delivery to treat laryngeal cancer. In the present study, novel bionanocomposites containingmore » nutrient agar coated gold nano particles (N-AuNPs) coupled with 6-mercaptopurine (drug) (N-AuNPs-Mp), folic acid (ligand) (N-AuNPs-Mp-Fa) and rhodamine (dye) (N-AuNPs-Rd), a fluorescent agent, were prepared and characterized by IR, UV, TEM, Particle size analysis and in vitro stability. The toxicity and fluorescence of N-Au was studied using zebrafish embryo model. The in vitro cytotoxicity of free Mp, N-Au-Mp and N-Au-Mp-Fa against HEp-2 cells was compared and found that the amount of Mp required to achieve 50% of growth of inhibition (IC{sub 50}) was much lower in N-Au-Mp-Fa than in free Mp and N-Au-Mp.« less

Off-line solid phase extraction and liquid chromatography-tandem mass spectrometry method for the quantitation of brivaracetam acid metabolites: Method validation and application to in vitro metabolism assays.

PubMed

Bourgogne, Emmanuel; Culot, Benoit; Dell'Aiera, Sylvie; Chanteux, Hugues; Stockis, Armel; Nicolas, Jean-Marie

2018-06-01

Brivaracetam (BRV) is a new high affinity synaptic vesicle protein 2A ligand recently approved for adults with partial-onset seizures. As a support to in vitro metabolism assays, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled to off-line solid phase extraction (SPE) was developed to quantify BRV acid metabolites, that is, BRV-AC (carboxylic derivative derived from BRV hydrolysis) and BRV-OHAC (corresponding to hydroxylated BRV-AC). The method was validated for various incubates (liver and kidney tissue homogenates and blood, all from humans) and applied to in vitro metabolism assays. The analytes were isolated from buffered samples using ISOLUTE C8 96-well SPE plates. Chromatographic separation was achieved on a Waters Atlantis T3 C18 analytical column (2.1 mm × 50 mm, 5 μm) with detection accomplished using a Waters Premier tandem mass spectrometer in positive ion electrospray and multiple reaction monitoring (MRM) mode. The standard curves, which ranged from 1.00 to 200 ng/mL for BRV-AC, BRV-OHAC, were fitted to a 1/x 2 weighted linear regression model. The intra-assay precision and inter-assay precision (expressed as coefficient of variation -%CV) were <8.5%, and the assay accuracy (deviation - %Dev) was within ±7.1% for the different matrices. This accurate, precise, and selective SPE/LC-MS/MS method has been successfully applied to in vitro assays aimed at characterizing the kinetics of BRV hydrolysis. BRV was found to be a better substrate for hydrolysis than its hydroxylated metabolite BRV-OH. BRV hydrolysis was detected in blood, liver and kidneys, demonstrating the broad distribution of the enzyme catalyzing the reaction. Copyright © 2018 Elsevier B.V. All rights reserved.

Mechanical testing of bones: the positive synergy of finite-element models and in vitro experiments.

PubMed

Cristofolini, Luca; Schileo, Enrico; Juszczyk, Mateusz; Taddei, Fulvia; Martelli, Saulo; Viceconti, Marco

2010-06-13

Bone biomechanics have been extensively investigated in the past both with in vitro experiments and numerical models. In most cases either approach is chosen, without exploiting synergies. Both experiments and numerical models suffer from limitations relative to their accuracy and their respective fields of application. In vitro experiments can improve numerical models by: (i) preliminarily identifying the most relevant failure scenarios; (ii) improving the model identification with experimentally measured material properties; (iii) improving the model identification with accurately measured actual boundary conditions; and (iv) providing quantitative validation based on mechanical properties (strain, displacements) directly measured from physical specimens being tested in parallel with the modelling activity. Likewise, numerical models can improve in vitro experiments by: (i) identifying the most relevant loading configurations among a number of motor tasks that cannot be replicated in vitro; (ii) identifying acceptable simplifications for the in vitro simulation; (iii) optimizing the use of transducers to minimize errors and provide measurements at the most relevant locations; and (iv) exploring a variety of different conditions (material properties, interface, etc.) that would require enormous experimental effort. By reporting an example of successful investigation of the femur, we show how a combination of numerical modelling and controlled experiments within the same research team can be designed to create a virtuous circle where models are used to improve experiments, experiments are used to improve models and their combination synergistically provides more detailed and more reliable results than can be achieved with either approach singularly.

A preliminary study for the development and optimization by experimental design of an in vitro method for prediction of drug buccal absorption.

PubMed

Mura, Paola; Orlandini, Serena; Cirri, Marzia; Maestrelli, Francesca; Mennini, Natascia; Casella, Giada; Furlanetto, Sandra

2018-06-15

The work was aimed at developing an in vitro method able to provide rapid and reliable evaluation of drug absorption through buccal mucosa. Absorption simulator apparatus endowed with an artificial membrane was purposely developed by experimental design. The apparent permeation coefficient (P app ) through excised porcine buccal mucosa of naproxen, selected as model drug, was the target value to obtain with the artificial membrane. The multivariate approach enabled systematic evaluation of the effect on the response (P app ) of simultaneous variations of the variables (kind of lipid components for support impregnation and relative amounts). A screening phase followed by a response-surface study allowed optimization of the lipid-mixture composition to obtain the desired P app value, and definition of a design space where all mixture components combinations fulfilled the desired target at a fixed probability level. The method offers a useful tool for a quick screening in the early stages of drug discovery and/or in preformulation studies, improving efficiency and chance of success in the development of buccal delivery systems. Further studies with other model drugs are planned to confirm the buccal absorption predictive capacity of the developed membrane. Copyright © 2018 Elsevier B.V. All rights reserved.

Modeling growth and dissemination of lymphoma in a co-evolving lymph node: a diffuse-domain approach

NASA Astrophysics Data System (ADS)

Chuang, Yao-Li; Cristini, Vittorio; Chen, Ying; Li, Xiangrong; Frieboes, Hermann; Lowengrub, John

2013-03-01

While partial differential equation models of tumor growth have successfully described various spatiotemporal phenomena observed for in-vitro tumor spheroid experiments, one challenge towards taking these models to further study in-vivo tumors is that instead of relatively static tissue culture with regular boundary conditions, in-vivo tumors are often confined in organ tissues that co-evolve with the tumor growth. Here we adopt a recently developed diffuse-domain method to account for the co-evolving domain boundaries, adapting our previous in-vitro tumor model for the development of lymphoma encapsulated in a lymph node, which may swell or shrink due to proliferation and dissemination of lymphoma cells and treatment by chemotherapy. We use the model to study the induced spatial heterogeneity, which may arise as an emerging phenomenon in experimental observations and model analysis. Spatial heterogeneity is believed to lead to tumor infiltration patterns and reduce the efficacy of chemotherapy, leaving residuals that cause cancer relapse after the treatment. Understanding the spatiotemporal evolution of in-vivo tumors can be an essential step towards more effective strategies of curing cancer. Supported by NIH-PSOC grant 1U54CA143907-01.

Three-Dimensional Human Tissue Models That Incorporate Diabetic Foot Ulcer-Derived Fibroblasts Mimic In Vivo Features of Chronic Wounds

PubMed Central

Maione, Anna G.; Brudno, Yevgeny; Stojadinovic, Olivera; Park, Lara K.; Smith, Avi; Tellechea, Ana; Leal, Ermelindo C.; Kearney, Cathal J.; Veves, Aristidis; Tomic-Canic, Marjana; Mooney, David J.

2015-01-01

Diabetic foot ulcers (DFU) are a major, debilitating complication of diabetes mellitus. Unfortunately, many DFUs are refractory to existing treatments and frequently lead to amputation. The development of more effective therapies has been hampered by the lack of predictive in vitro methods to investigate the mechanisms underlying impaired healing. To address this need for realistic wound-healing models, we established patient-derived fibroblasts from DFUs and site-matched controls and used them to construct three-dimensional (3D) models of chronic wound healing. Incorporation of DFU-derived fibroblasts into these models accurately recapitulated the following key aspects of chronic ulcers: reduced stimulation of angiogenesis, increased keratinocyte proliferation, decreased re-epithelialization, and impaired extracellular matrix deposition. In addition to reflecting clinical attributes of DFUs, the wound-healing potential of DFU fibroblasts demonstrated in this suite of models correlated with in vivo wound closure in mice. Thus, the reported panel of 3D DFU models provides a more biologically relevant platform for elucidating the cell–cell and cell–matrix-related mechanisms responsible for chronic wound pathogenesis and may improve translation of in vitro findings into efficacious clinical applications. PMID:25343343

Performance of disposable endoscopic forceps according to the manufacturing techniques.

PubMed

Kwon, Chang-Il; Kim, Gwangil; Moon, Jong Pil; Yun, Ho; Ko, Weon Jin; Cho, Joo Young; Hong, Sung Pyo

2018-03-05

Recently, to lower the production costs and risk of infection, new disposable biopsy forceps made using simple manufacturing techniques have been introduced. However, the effects of the manufacturing techniques are unclear. The aim of this study was to evaluate which types of biopsy forceps could obtain good-quality specimens according to the manufacturing techniques. By using an in vitro nitrile glove popping model, we compared the popping ability among eight different disposable biopsy forceps (one pair of biopsy forceps with cups made by a cutting method [cutting forceps], four pairs of biopsy forceps with cups made by a pressing method [pressing forceps], and three pairs of biopsy forceps with cups made using a injection molding method [molding forceps]). Using an in vivo swine model, we compared the penetration depth and quality of specimen among the biopsy forceps. In the in vitro model, the molding forceps provided a significantly higher popping rate than the other forceps (cutting forceps, 25.0%; pressing forceps, 17.5%; and molding forceps, 41.7%; p = 0.006). In the in vivo model, the cutting and pressing forceps did not provide larger specimens, deeper biopsy specimen, and higher specimen adequacy than those obtained using the molding forceps (p = 0.2631, p = 0.5875, and p = 0.2147, respectively). However, the molding forceps showed significantly more common crush artifact than the others (cutting forceps, 0%; pressing forceps, 5.0%; and molding forceps, 43.3%; p = 0.0007). The molding forceps provided lower performance than the cutting and pressing forceps in terms of crush artifact.

Design and Characterization of a Microfabricated Hydrogen Clearance Blood Flow Sensor

PubMed Central

Walton, Lindsay R.; Edwards, Martin A.; McCarty, Gregory S.; Wightman, R. Mark

2016-01-01

Background Modern cerebral blood flow (CBF) detection favors the use of either optical technologies that are limited to cortical brain regions, or expensive magnetic resonance. Decades ago, inhalation gas clearance was the choice method of quantifying CBF, but this suffered from poor temporal resolution. Electrolytic H2 clearance (EHC) generates and collects gas in situ at an electrode pair, which improves temporal resolution, but the probe size has prohibited meaningful subcortical use. New Method We microfabricated EHC electrodes to an order of magnitude smaller than those existing, on the scale of 100 µm, to permit use deep within the brain. Results Novel EHC probes were fabricated. The devices offered exceptional signal-to-noise, achieved high collection efficiencies (40 – 50%) in vitro, and agreed with theoretical modeling. An in vitro chemical reaction model was used to confirm that our devices detected flow rates higher than those expected physiologically. Computational modeling that incorporated realistic noise levels demonstrated devices would be sensitive to physiological CBF rates. Comparison with Existing Method The reduced size of our arrays makes them suitable for subcortical EHC measurements, as opposed to the larger, existing EHC electrodes that would cause substantial tissue damage. Our array can collect multiple CBF measurements per minute, and can thus resolve physiological changes occurring on a shorter timescale than existing gas clearance measurements. Conclusion We present and characterize microfabricated EHC electrodes and an accompanying theoretical model to interpret acquired data. Microfabrication allows for the high-throughput production of reproducible devices that are capable of monitoring deep brain CBF with sub-minute resolution. PMID:27102042

Evaluation of fibrin-based dermal-epidermal organotypic cultures for in vitro skin corrosion and irritation testing of chemicals according to OECD TG 431 and 439.

PubMed

Morales, Mariana; Pérez, David; Correa, Luis; Restrepo, Luz

2016-10-01

Reconstructed human epidermis (RhE) models have been used for in vitro testing of the potential harmful effects of exposure to chemical compounds on health. In the past, skin irritation and corrosion were evaluated in animal models; however, in recent years, due to the bioethics implications of the method and, to minimize the use of experimental animals, alternative procedures have been proposed. The Organisation for Economic Co-operation and Development (OECD) in its test guidelines (TG) 431 and 439 indicates the requirements for validating new methods for the evaluation of skin corrosion and irritation, respectively. Here, we present an in-house human dermal-epidermal model, useful for the performance of these tests. Using the methods described in this work, it was possible to obtain human fibrin-based dermal-epidermal organotypic skin cultures (ORGs) displaying similar histological characteristics to native skin and expressing specific differentiation epithelial proteins. The end points to classify a substance as irritant or corrosive were cell viability evaluated by MTT assay, and cytokine release measured by BD CBA for human inflammatory cytokines. According to the MTT test, the ORGs correctly classified irritating and corrosive substances. Moreover, the cytokine release assay was difficult to interpret in the context of testing chemical hazard classification. Further experiments are needed to validate this new model for the evaluation of surfactants because the fibrin matrix was affected in the presence of these substances. Copyright © 2016 Elsevier B.V. All rights reserved.

ITC Recommendations for Transporter Kinetic Parameter Estimation and Translational Modeling of Transport-Mediated PK and DDIs in Humans

PubMed Central

Zamek-Gliszczynski, MJ; Lee, CA; Poirier, A; Bentz, J; Chu, X; Ellens, H; Ishikawa, T; Jamei, M; Kalvass, JC; Nagar, S; Pang, KS; Korzekwa, K; Swaan, PW; Taub, ME; Zhao, P; Galetin, A

2013-01-01

This white paper provides a critical analysis of methods for estimating transporter kinetics and recommendations on proper parameter calculation in various experimental systems. Rational interpretation of transporter-knockout animal findings and application of static and dynamic physiologically based modeling approaches for prediction of human transporter-mediated pharmacokinetics and drug–drug interactions (DDIs) are presented. The objective is to provide appropriate guidance for the use of in vitro, in vivo, and modeling tools in translational transporter science. PMID:23588311

[Analysis on microdialysis probe recovery of baicalin in vitro and in vivo based on LC-MS/MS].

PubMed

Chen, Teng-Fei; Liu, Jian-Xun; Zhang, Ying; Lin, Li; Song, Wen-Ting; Yao, Ming-Jiang

2017-06-01

To further study the brain behavior and the pharmacokinetics of baicalin in intercellular fluid of brain, and study the recovery rate and stability of brain and blood microdialysis probe of baicalin in vitro and in vivo. The concentration of baicalin in brain and blood microdialysates was determined by LC-MS/MS and the probe recovery for baicalin was calculated. The effects of different flow rates (0.50, 1.0, 1.5, 2.0，3.0 μL•min⁻¹) on recovery in vitro were determined by incremental method and decrement method. The effects of different drug concentrations (50.00, 200.0, 500.0, 1 000 μg•L⁻¹) and using times (0, 1, 2) on recovery in vitro were determined by incremental method. The probe recovery stability and effect of flow rate on recovery in vivo were determined by decrement method, and its results were compared with those in in vitro trial. The in vitro recovery of brain and blood probe of baicalin was decreased with the increase of flow rate under the same concentration; and at the same flow rate, different concentrations of baicalin had little influence on the recovery. The probe which had been used for 2 times showed no obvious change in probe recovery by syringe with 2% heparin sodium and ultrapure water successively. In vitro recovery rates obtained by incremental method and decrement method were approximately equal under the same condition, and the in vivo recovery determined by decrement method was similar with the in vitro results and they were showed a good stability within 10 h. The results showed that decrement method can be used for pharmacokinetic study of baicalin, and can be used to study probe recovery in vivo at the same time. Copyright© by the Chinese Pharmaceutical Association.

Accelerated fatigue testing of dentin-composite bond with continuously increasing load.

PubMed

Li, Kai; Guo, Jiawen; Li, Yuping; Heo, Young Cheul; Chen, Jihua; Xin, Haitao; Fok, Alex

2017-06-01

The aim of this study was to evaluate an accelerated fatigue test method that used a continuously increasing load for testing the dentin-composite bond strength. Dentin-composite disks (ϕ5mm×2mm) made from bovine incisor roots were subjected to cyclic diametral compression with a continuously increasingly load amplitude. Two different load profiles, linear and nonlinear with respect to the number of cycles, were considered. The data were then analyzed by using a probabilistic failure model based on the Weakest-Link Theory and the classical stress-life function, before being transformed to simulate clinical data of direct restorations. All the experimental data could be well fitted with a 2-parameter Weibull function. However, a calibration was required for the effective stress amplitude to account for the difference between static and cyclic loading. Good agreement was then obtained between theory and experiments for both load profiles. The in vitro model also successfully simulated the clinical data. The method presented will allow tooth-composite interfacial fatigue parameters to be determined more efficiently. With suitable calibration, the in vitro model can also be used to assess composite systems in a more clinically relevant manner. Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Role of In Vitro Release Methods in Liposomal Formulation Development: Challenges and Regulatory Perspective.

PubMed

Solomon, Deepak; Gupta, Nilesh; Mulla, Nihal S; Shukla, Snehal; Guerrero, Yadir A; Gupta, Vivek

2017-11-01

In the past few years, measurement of drug release from pharmaceutical dosage forms has been a focus of extensive research because the release profile obtained in vitro can give an indication of the drug's performance in vivo. Currently, there are no compendial in vitro release methods designed for liposomes owing to a range of experimental challenges, which has created a major hurdle for both development and regulatory acceptance of liposome-based drug products. In this paper, we review the current techniques that are most often used to assess in vitro drug release from liposomal products; these include the membrane diffusion techniques (dialysis, reverse dialysis, fractional dialysis, and microdialysis), the sample-and-separate approach, the in situ method, the continuous flow, and the modified United States Pharmacopeia methods (USP I and USP IV). We discuss the principles behind each of the methods and the criteria that assist in choosing the most appropriate method for studying drug release from a liposomal formulation. Also, we have included information concerning the current regulatory requirements for liposomal drug products in the United States and in Europe. In light of increasing costs of preclinical and clinical trials, applying a reliable in vitro release method could serve as a proxy to expensive in vivo bioavailability studies. Graphical Abstract Appropriate in-vitro drug release test from liposomal products is important to predict the in-vivo performance.

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

PubMed Central

Shipley, Mackenzie M.; Mangold, Colleen A.; Szpara, Moriah L.

2016-01-01

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease. PMID:26967710

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line.

PubMed

Shipley, Mackenzie M; Mangold, Colleen A; Szpara, Moriah L

2016-02-17

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods(1-4) and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

In Vitro and Modeling Approaches to Risk Assessment from the U.S. Environmental Protection Agency ToxCast Program

EPA Science Inventory

A significant challenge in toxicology is the “too many chemicals” problem. Humans and environmental species are exposed to as many as tens of thousands of chemicals, only a small percentage of which have been tested thoroughly using standard in vivo test methods. This paper revie...

Using ToxCast in vitro Assays in the Hierarchical Quantitative Structure-Activity Relationship (QSAR) Modeling for Predicting in vivo Toxicity of Chemicals

EPA Science Inventory

The goal of chemical toxicology research is utilizing short term bioassays and/or robust computational methods to predict in vivo toxicity endpoints for chemicals. The ToxCast program established at the US Environmental Protection Agency (EPA) is addressing this goal by using ca....

Application of the ToxMiner Database: Network Analysis of Linkage between ToxCast Phase I Chemicals and Thyroid Related Disease Outcomes

EPA Science Inventory

The US EPA ToxCast program is using in vitro HTS (High-Throughput Screening) methods to profile and model bioactivity of environmental chemicals. The main goals of the ToxCast program are to generate predictive signatures of toxicity, and ultimately provide rapid and cost-effecti...

Comparison of in situ and in vitro techniques for measuring ruminal degradation of animal by-product proteins.

PubMed

England, M L; Broderick, G A; Shaver, R D; Combs, D K

1997-11-01

Ruminally undegraded protein (RUP) values of blood meal (n = 2), hydrolyzed feather meal (n = 2), fish meal (n = 2), meat and bone meal, and soybean meal were estimated using an in situ method, an inhibitor in vitro method, and an inhibitor in vitro technique applying Michaelis-Menten saturation kinetics. Degradation rates for in situ and inhibitor in vitro methods were calculated by regression of the natural log of the proportion of crude protein (CP) remaining undegraded versus time. Nonlinear regression analysis of the integrated Michaelis-Menten equation was used to determine maximum velocity, the Michaelis constant, and degradation rate (the ratio of maximum velocity to the Michaelis constant). A ruminal passage rate of 0.06/h was assumed in the calculation of RUP. The in situ and inhibitor in vitro techniques yielded similar estimates of ruminal degradation. Mean RUP estimated for soybean meal, blood meal, hydrolyzed feather meal, fish meal, and meat and bone meal were, respectively, 28.6, 86.0, 77.4, 52.9, and 52.6% of CP by the in situ method and 26.4, 86.1, 76.0, 59.6, and 49.5% of CP by the inhibitor in vitro technique. The Michaelis-Menten inhibitor in vitro technique yielded more rapid CP degradation rates and decreased estimates of RUP. The inhibitor in vitro method required less time and labor than did the other two techniques to estimate the RUP values of animal by-product proteins. Results from in vitro incubations with pepsin.HCl suggested that low postruminal digestibility of hydrolyzed feather meal may impair its value as a source of RUP.

Evaluation of clay/poly (L-lactide) microcomposites as anticancer drug, 6-mercaptopurine reservoir through in vitro cytotoxicity, oxidative stress markers and in vivo pharmacokinetics.

PubMed

Kevadiya, Bhavesh D; Chettiar, Shiva Shankaran; Rajkumar, Shalini; Bajaj, Hari C; Gosai, Kalpeshgiri A; Brahmbhatt, Harshad

2013-12-01

Intercalation of 6-mercaptopurine (6-MP), an antineoplastic drug in interlayer gallery of Na(+)-clay (MMT) was further entrapped in poly (L-lactide) matrix to form microcomposite spheres (MPs) in order to reduce the cell toxicity and enhance in vitro release and pharmacokinetic proficiency. The drug-clay hybrid was fabricated via intercalation by ion-exchange method to form MPs from hybrid. In vitro drug release showed controlled pattern, fitted to kinetic models suggested controlled exchange and partial diffusion through swollen matrix of clay inter layered gallery. The in vitro efficacy of formulated composites drug was tested in Human neuroblastoma cell line (IMR32) by various cell cytotoxic and oxidative stress marker indices. In vivo pharmacokinetics suggested that the intensity of formulated drug level in plasma was within remedial borders as compared to free drug. These clay based composites therefore have great potential of becoming a new dosage form of 6-MP. Copyright © 2013 Elsevier B.V. All rights reserved.

Insights and Perspectives on Emerging Inputs to Weight of Evidence Determinations for Food Safety: Workshop Proceedings

PubMed Central

Bialk, Heidi; Llewellyn, Craig; Kretser, Alison; Canady, Richard; Lane, Richard; Barach, Jeffrey

2013-01-01

This workshop aimed to elucidate the contribution of computational and emerging in vitro methods to the weight of evidence used by risk assessors in food safety assessments. The following issues were discussed: using in silico and high-throughput screening (HTS) data to confirm the safety of approved food ingredients, applying in silico and HTS data in the process of assessing the safety of a new food ingredient, and utilizing in silico and HTS data in communicating the safety of food ingredients while enhancing the public’s trust in the food supply. Perspectives on integrating computational modeling and HTS assays as well as recommendations for optimizing predictive methods for risk assessment were also provided. Given the need to act quickly or proceed cautiously as new data emerge, this workshop also focused on effectively identifying a path forward in communicating in silico and in vitro data. PMID:24296863

COMPARISON OF IN VIVO AND IN VITRO CHEMICAL SCREENING ASSAYS FOR ESTROGEN-RESPONSIVE PROTEIN BIOMARKER EXPRESSION IN THE SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS)

EPA Science Inventory

Animal welfare concerns and the high cost associated with in vivo chemical screening methods have prompted efforts to develop highly specific and sensitive in vitro techniques to evaluate chemicals for endocrine activity. Diagnostic biomarkers developed using in vitro methods sho...

An in vitro screening method to evaluate chemicals as potential chemotherapeutants to control Aeromonas hydrophila infection in channel catfish

USDA-ARS?s Scientific Manuscript database

Using catfish gill cells G1B and four chemicals (hydrogen peroxide, sodium chloride, potassium permanganate, and D-mannose), the feasibility of using an in vitro screening method to identify potential effective chemotherapeutants was evaluated in this study. In vitro screening results revealed that,...

Time-controlled release pseudoephedrine tablets: bioavailability and in vitro/in vivo correlations.

PubMed

Halsas, M; Penttinen, T; Veski, P; Jürjenson, H; Marvola, M

2001-09-01

In chronopharmacotherapy, circadian changes in disease symptoms are taken into account. Press-coated, time-controlled release tablets containing pseudoephedrine hydrochloride as a model drug have been formulated and the suitability of this highly soluble drug in relation to the new drug delivery system was evaluated. Hydroxypropylmethylcellulose was used in the coat of the tablet to adjust drug release. If such a formulation was administered in the evening it would have maximal effect in the early morning, and would be useful for the treatment of nocturnal symptoms. Two cross-over, single-dose bioavailability studies were carried out on eight healthy volunteers. A dissolution test method was developed to establish level A and level C in vitro/in vivo correlation for four formulations. With a low viscosity grade of polymer, peak concentrations were achieved after five hours. The drug was absorbed much more slowly from tablets containing a high viscosity grade polymer, with a plasma peak at ten hours. For further development of the drug delivery system described, a dissolution test method at pH 7.2 at a rotation speed of 150 min-1 is recommended on the basis of level A in vitro/in vivo correlation.

Dynamic PET and Optical Imaging and Compartment Modeling using a Dual-labeled Cyclic RGD Peptide Probe

PubMed Central

Zhu, Lei; Guo, Ning; Li, Quanzheng; Ma, Ying; Jacboson, Orit; Lee, Seulki; Choi, Hak Soo; Mansfield, James R.; Niu, Gang; Chen, Xiaoyuan

2012-01-01

Purpose: The aim of this study is to determine if dynamic optical imaging could provide comparable kinetic parameters to that of dynamic PET imaging by a near-infrared dye/64Cu dual-labeled cyclic RGD peptide. Methods: The integrin αvβ3 binding RGD peptide was conjugated with a macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for copper labeling and PET imaging and a near-infrared dye ZW-1 for optical imaging. The in vitro biological activity of RGD-C(DOTA)-ZW-1 was characterized by cell staining and receptor binding assay. Sixty-min dynamic PET and optical imaging were acquired on a MDA-MB-435 tumor model. Singular value decomposition (SVD) method was applied to compute the dynamic optical signal from the two-dimensional optical projection images. Compartment models were used to quantitatively analyze and compare the dynamic optical and PET data. Results: The dual-labeled probe 64Cu-RGD-C(DOTA)-ZW-1 showed integrin specific binding in vitro and in vivo. The binding potential (Bp) derived from dynamic optical imaging (1.762 ± 0.020) is comparable to that from dynamic PET (1.752 ± 0.026). Conclusion: The signal un-mixing process using SVD improved the accuracy of kinetic modeling of 2D dynamic optical data. Our results demonstrate that 2D dynamic optical imaging with SVD analysis could achieve comparable quantitative results as dynamic PET imaging in preclinical xenograft models. PMID:22916074

In vitro methods for hazard assessment of industrial chemicals – opportunities and challenges

PubMed Central

Wong, Chin Lin; Ghassabian, Sussan; Smith, Maree T.; Lam, Ai-Leen

2015-01-01

Allergic contact dermatitis (ACD) is a delayed-type hypersensitivity immune reaction mediated by T-lymphocytes as a result of repeated exposure of an allergen primarily on skin. ACD accounts for up to 95% of occupational skin diseases, with epoxy resins implicated as one of the most common causes of ACD. Efficient high-throughput in vitro screening for accurate identification of compounds and materials that may pose hazardous risks in the workplace is crucial. At present, the murine local lymph node assay is the ‘method of choice’ for predicting the sensitizing potency of contact allergens. As the 3Rs principles of reduction, refinement, and replacement in animal testing has gained political and economic momentum, several in vitro screening methods have been developed for identifying potential contact allergens. To date, these latter methods have been utilized primarily to assess the skin sensitizing potential of the chemical components of cosmetic products with scant research attention as to the applicability of these methods to industrial chemicals, particularly epoxy resins. Herein we review the currently utilized in vitro methods and identify the knowledge gaps with regard to assessing the generalizability of in vitro screening methods for assessing the skin sensitizing potential of industrial chemicals. PMID:25999858

In vitro methods for hazard assessment of industrial chemicals - opportunities and challenges.

PubMed

Wong, Chin Lin; Ghassabian, Sussan; Smith, Maree T; Lam, Ai-Leen

2015-01-01

Allergic contact dermatitis (ACD) is a delayed-type hypersensitivity immune reaction mediated by T-lymphocytes as a result of repeated exposure of an allergen primarily on skin. ACD accounts for up to 95% of occupational skin diseases, with epoxy resins implicated as one of the most common causes of ACD. Efficient high-throughput in vitro screening for accurate identification of compounds and materials that may pose hazardous risks in the workplace is crucial. At present, the murine local lymph node assay is the 'method of choice' for predicting the sensitizing potency of contact allergens. As the 3Rs principles of reduction, refinement, and replacement in animal testing has gained political and economic momentum, several in vitro screening methods have been developed for identifying potential contact allergens. To date, these latter methods have been utilized primarily to assess the skin sensitizing potential of the chemical components of cosmetic products with scant research attention as to the applicability of these methods to industrial chemicals, particularly epoxy resins. Herein we review the currently utilized in vitro methods and identify the knowledge gaps with regard to assessing the generalizability of in vitro screening methods for assessing the skin sensitizing potential of industrial chemicals.

Prediction of in vitro and in vivo oestrogen receptor activity using hierarchical clustering

EPA Science Inventory

In this study, hierarchical clustering classification models were developed to predict in vitro and in vivo oestrogen receptor (ER) activity. Classification models were developed for binding, agonist, and antagonist in vitro ER activity and for mouse in vivo uterotrophic ER bindi...

Evaluating drug efficacy and toxicology in three dimensions: using synthetic extracellular matrices in drug discovery.

PubMed

Prestwich, Glenn D

2008-01-01

The acceptance of the new paradigm of 3-D cell culture is currently constrained by the lack of a biocompatible material in the marketplace that offers ease of use, experimental flexibility, and a seamless transition from in vitro to in vivo applications. I describe the development of a covalently cross-linked mimic of the extracellular matrix (sECM), now commercially available, for 3-D culture of cells in vitro and for translational use in vivo. These bio-inspired, biomimetic materials can be used "as is" in drug discovery, toxicology, cell banking, and, ultimately, medicine. For cell therapy and the development of clinical combination products, the sECM biomaterials must be highly reproducible, manufacturable, approvable, and affordable. To obtain integrated, functional, multicellular systems that recapitulate tissues and organs, the needs of the true end users, physicians and patients, must dictate the key design criteria. In chemical terms, the sECM consists of chemically-modified hyaluronan (HA), other glycosaminoglycans (GAGs), and ECM polypeptides containing thiol residues that are cross-linked using biocompatible polyvalent electrophiles. For example, co-cross-linking the semisynthetic thiol-modified HA-like GAG with thiol-modified gelatin produces Extracel as a hydrogel. This hydrogel may be formed in situ in the presence of cells or tissues to provide an injectable cell-delivery vehicle. Alternately, an Extracel hyrogel can be lyophilized to create a macroporous scaffold, which can then be employed for 3-D cell culture. In this Account, we describe four applications of sECMs that are relevant to the evaluation of drug efficacy and drug toxicity. First, the uses of sECMs to promote both in vitro and in vivo growth of healthy cellularized 3-D tissues are summarized. Primary or cell-line-derived cells, including fibroblasts, chondrocytes, hepatocytes, adult and embryonic stem cells, and endothelial and epithelial cells have been used. Second, primary hepatocytes retain their biochemical phenotypes and achieve greater longevity in 3-D culture in Extracel. This constitutes a new 3-D method for rapid evaluation of hepatotoxicity in vitro. Third, cancer cell lines are readily grown in 3-D culture in Extracel, offering a method for rapid evaluation of new anticancer agents in a more physiological ex vivo tumor model. This system has been used to evaluate signal transduction modifiers obtained from our research on lipid signaling. Fourth, a new "tumor engineering" xenograft model uses orthotopic injection of Extracel-containing tumor cells in nude mice. This approach allows production of patient-specific mice using primary human tumor samples and offers a superior metastatic cancer model. Future applications of the injectable cell delivery and 3-D cell culture methods include chemoattractant and angiogenesis assays, high-content automated screening of chemical libraries, pharmacogenomic and toxicogenomic studies with cultured organoids, and personalized treatment models. In summary, the sECM technology offers a versatile "translational bridge" from in vitro to in vivo to facilitate drug discovery in both academic and pharmaceutical laboratories.

Low-Intensity Pulsed Ultrasound Protects Retinal Ganglion Cell From Optic Nerve Injury Induced Apoptosis via Yes Associated Protein

PubMed Central

Zhou, Jia-Xing; Liu, Yun-Jia; Chen, Xi; Zhang, Xi; Xu, Jie; Yang, Ke; Wang, Dong; Lin, Sen; Ye, Jian

2018-01-01

Background: Low-intensity pulsed ultrasound (LIPUS) has been used in clinical studies. But little is known about its effects on the central nervous system (CNS), or its mechanism of action. Retinal ganglion cells (RGCs) are CNS neuronal cells that can be utilized as a classic model system to evaluate outcomes of LIPUS protection from external trauma-induced retinal injury. In this study, we aim to: (1) determine the pulse energy and the capability of LIPUS in RGC viability, (2) ascertain the protective role of LIPUS in optic nerve (ON) crush-induced retinal injury, and 3) explore the cellular mechanisms of RGC apoptosis prevention by LIPUS. Methods: An ON crush model was set up to induce RGC death. LIPUS was used to treat mice eyes daily, and the retina samples were dissected for immunostaining and Western blot. The expression of yes-associated protein (YAP) and apoptosis-related proteins was detected by immunostaining and Western blot in vitro and in vivo. Apoptosis of RGCs was evaluated by TUNEL staining, the survival of RGCs and retained axons were labeled by Fluoro-gold and Tuj1 antibody, respectively. Rotenone was used to set up an in vitro cellular degenerative model and siYAP was used to interfering the expression of YAP to detect the LIPUS protective function. Results: LIPUS protected RGC from loss and apoptosis in vivo and in vitro. The ratio of cleaved/pro-caspase3 also decreased significantly under LIPUS treatment. As a cellular mechanical sensor, YAP expression increased and YAP translocated to nucleus in LIPUS stimulation group, however, phospho-YAP was found to be decreased. When YAP was inhibited, the LIPUS could not protect RGC from caspase3-dependent apoptosis. Conclusion: LIPUS prevented RGCs from apoptosis in an ON crush model and in vitro cellular degenerative model, which indicates a potential treatment for further traumatic ON injury. The mechanism of protection is dependent on YAP activation and correlated with caspase-3 signaling.

Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells

PubMed Central

Jeon, Hye Lyun; Yi, Jung-Sun; Kim, Tae Sung; Oh, Youkyung; Lee, Hye Jeong; Lee, Minseong; Bang, Jin Seok; Ko, Kinarm; Ahn, Il Young; Ko, Kyungyuk; Kim, Joohwan; Park, Hye-Kyung; Lee, Jong Kwon; Sohn, Soo Jung

2017-01-01

Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration (IC50) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity. PMID:28443181

Investigation of metabolic objectives in cultured hepatocytes.

PubMed

Uygun, Korkut; Matthew, Howard W T; Huang, Yinlun

2007-06-15

Using optimization based methods to predict fluxes in metabolic flux balance models has been a successful approach for some microorganisms, enabling construction of in silico models and even inference of some regulatory motifs. However, this success has not been translated to mammalian cells. The lack of knowledge about metabolic objectives in mammalian cells is a major obstacle that prevents utilization of various metabolic engineering tools and methods for tissue engineering and biomedical purposes. In this work, we investigate and identify possible metabolic objectives for hepatocytes cultured in vitro. To achieve this goal, we present a special data-mining procedure for identifying metabolic objective functions in mammalian cells. This multi-level optimization based algorithm enables identifying the major fluxes in the metabolic objective from MFA data in the absence of information about critical active constraints of the system. Further, once the objective is determined, active flux constraints can also be identified and analyzed. This information can be potentially used in a predictive manner to improve cell culture results or clinical metabolic outcomes. As a result of the application of this method, it was found that in vitro cultured hepatocytes maximize oxygen uptake, coupling of urea and TCA cycles, and synthesis of serine and urea. Selection of these fluxes as the metabolic objective enables accurate prediction of the flux distribution in the system given a limited amount of flux data; thus presenting a workable in silico model for cultured hepatocytes. It is observed that an overall homeostasis picture is also emergent in the findings.

Flavonoids from Potentilla parvifolia Fisch. and Their Neuroprotective Effects in Human Neuroblastoma SH-SY5Y Cells in vitro.

PubMed

Yuan, Zhenzhen; Luan, Guangxiang; Wang, Zhenhua; Hao, Xueyan; Li, Ji; Suo, Yourui; Li, Gang; Wang, Honglun

2017-06-01

Potentilla parvifolia Fisch. (Rosaceae) is a traditional medicinal plant in P. R. China. In this study, seven flavonoids, ayanin (1), tricin (2), quercetin (3), tiliroside (4), miquelianin (5), isoquercitrin (6), and astragalin (7), were separated and purified from ethyl acetate extractive fractions from ethanol extracts of P. parvifolia using a combination of sevaral chromatographic methods. The human neuroblastoma SH-SY5Y cells were differentiated with all trans-retinoic acid and treated with okadaic acid to induce tau protein phosphorylation and synaptic atrophy, which could establish an Alzheimer's disease cell model. The neuroprotective effects of these flavonoids in cellular were evaluated in vitro by this cell model. Results from the Western blot and morphology analysis suggested that compounds 3 and 4 had the better neuroprotective effects. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

Vibrational spectroscopy and imaging for concurrent cellular trafficking of co-localized doxorubicin and deuterated phospholipid vesicles

NASA Astrophysics Data System (ADS)

Misra, S. K.; Mukherjee, P.; Ohoka, A.; Schwartz-Duval, A. S.; Tiwari, S.; Bhargava, R.; Pan, D.

2016-01-01

Simultaneous tracking of nanoparticles and encapsulated payload is of great importance and visualizing their activity is arduous. Here we use vibrational spectroscopy to study the in vitro tracking of co-localized lipid nanoparticles and encapsulated drug employing a model system derived from doxorubicin-encapsulated deuterated phospholipid (dodecyl phosphocholine-d38) single tailed phospholipid vesicles.Simultaneous tracking of nanoparticles and encapsulated payload is of great importance and visualizing their activity is arduous. Here we use vibrational spectroscopy to study the in vitro tracking of co-localized lipid nanoparticles and encapsulated drug employing a model system derived from doxorubicin-encapsulated deuterated phospholipid (dodecyl phosphocholine-d38) single tailed phospholipid vesicles. Electronic supplementary information (ESI) available: Raman and confocal images of the Deuto-DOX-NPs in cells, materials and details of methods. See DOI: 10.1039/c5nr07975f

In vitro and ex vivo microbial leakage assessment in endodontics: A literature review.

PubMed

Savadkouhi, Sohrab Tour; Bakhtiar, Hengameh; Ardestani, Safoura Emami

2016-01-01

The aim of this study was to perform a literature review of published in-vitro and ex-vivo studies, which evaluated microbial leakage in endodontics in the past 10 years. A comprehensive electronic literature search was carried out in PubMed database for English articles published from 2005 to 2016 using the keywords "endodontics," " in vitro ," " ex vivo ," "microbial leakage," "microbial penetration," "saliva," " Enterococcus faecalis ," " E. faecalis ," "endodontic sealers," "temporary filling material," "apical plug," "mineral trioxide aggregate," and "MTA." The keywords were combined using Boolean operators AND/OR. Based on our search strategy, 33 relevant articles were included in the study. There are three main methods for assessment of bacterial microleakage, namely, (A) the dual-chamber leakage model, (B) detection of bacteria using a scanning electron microscope (SEM), and (C) polymerase chain reaction. All bacterial leakage models have some limitations and may yield different results compared to other microleakage evaluation techniques (i.e., dye penetration, fluid filtration, or electrochemical tests). The results of SEM correlated with those of microbial leakage test in most studies. Microbial leakage test using saliva better simulates the clinical setting for assessment of the leakage of single or mixed bacterial species.

Biological properties of solid free form designed ceramic scaffolds with BMP-2: in vitro and in vivo evaluation.

PubMed

Abarrategi, Ander; Moreno-Vicente, Carolina; Martínez-Vázquez, Francisco Javier; Civantos, Ana; Ramos, Viviana; Sanz-Casado, José Vicente; Martínez-Corriá, Ramón; Perera, Fidel Hugo; Mulero, Francisca; Miranda, Pedro; López-Lacomba, José Luís

2012-01-01

Porous ceramic scaffolds are widely studied in the tissue engineering field due to their potential in medical applications as bone substitutes or as bone-filling materials. Solid free form (SFF) fabrication methods allow fabrication of ceramic scaffolds with fully controlled pore architecture, which opens new perspectives in bone tissue regeneration materials. However, little experimentation has been performed about real biological properties and possible applications of SFF designed 3D ceramic scaffolds. Thus, here the biological properties of a specific SFF scaffold are evaluated first, both in vitro and in vivo, and later scaffolds are also implanted in pig maxillary defect, which is a model for a possible application in maxillofacial surgery. In vitro results show good biocompatibility of the scaffolds, promoting cell ingrowth. In vivo results indicate that material on its own conducts surrounding tissue and allow cell ingrowth, thanks to the designed pore size. Additional osteoinductive properties were obtained with BMP-2, which was loaded on scaffolds, and optimal bone formation was observed in pig implantation model. Collectively, data show that SFF scaffolds have real application possibilities for bone tissue engineering purposes, with the main advantage of being fully customizable 3D structures.

Biological Properties of Solid Free Form Designed Ceramic Scaffolds with BMP-2: In Vitro and In Vivo Evaluation

PubMed Central

Abarrategi, Ander; Moreno-Vicente, Carolina; Martínez-Vázquez, Francisco Javier; Civantos, Ana; Ramos, Viviana; Sanz-Casado, José Vicente; Martínez-Corriá, Ramón; Perera, Fidel Hugo; Mulero, Francisca; Miranda, Pedro; López-Lacomba, José Luís

2012-01-01

Porous ceramic scaffolds are widely studied in the tissue engineering field due to their potential in medical applications as bone substitutes or as bone-filling materials. Solid free form (SFF) fabrication methods allow fabrication of ceramic scaffolds with fully controlled pore architecture, which opens new perspectives in bone tissue regeneration materials. However, little experimentation has been performed about real biological properties and possible applications of SFF designed 3D ceramic scaffolds. Thus, here the biological properties of a specific SFF scaffold are evaluated first, both in vitro and in vivo, and later scaffolds are also implanted in pig maxillary defect, which is a model for a possible application in maxillofacial surgery. In vitro results show good biocompatibility of the scaffolds, promoting cell ingrowth. In vivo results indicate that material on its own conducts surrounding tissue and allow cell ingrowth, thanks to the designed pore size. Additional osteoinductive properties were obtained with BMP-2, which was loaded on scaffolds, and optimal bone formation was observed in pig implantation model. Collectively, data show that SFF scaffolds have real application possibilities for bone tissue engineering purposes, with the main advantage of being fully customizable 3D structures. PMID:22470527

Morphologic and molecular evaluation of Chlamydia trachomatis growth in human endocervix reveals distinct growth patterns

PubMed Central

Lewis, Maria E.; Belland, Robert J.; AbdelRahman, Yasser M.; Beatty, Wandy L.; Aiyar, Ashok A.; Zea, Arnold H.; Greene, Sheila J.; Marrero, Luis; Buckner, Lyndsey R.; Tate, David J.; McGowin, Chris L.; Kozlowski, Pamela A.; O'Brien, Michelle; Lillis, Rebecca A.; Martin, David H.; Quayle, Alison J.

2014-01-01

In vitro models of Chlamydia trachomatis growth have long been studied to predict growth in vivo. Alternative or persistent growth modes in vitro have been shown to occur under the influence of numerous stressors but have not been studied in vivo. Here, we report the development of methods for sampling human infections from the endocervix in a manner that permits a multifaceted analysis of the bacteria, host and the endocervical environment. Our approach permits evaluating total bacterial load, transcriptional patterns, morphology by immunofluorescence and electron microscopy, and levels of cytokines and nutrients in the infection microenvironment. By applying this approach to two pilot patients with disparate infections, we have determined that their contrasting growth patterns correlate with strikingly distinct transcriptional biomarkers, and are associated with differences in local levels of IFNγ. Our multifaceted approach will be useful to dissect infections in the human host and be useful in identifying patients at risk for chronic disease. Importantly, the molecular and morphological analyses described here indicate that persistent growth forms can be isolated from the human endocervix when the infection microenvironment resembles the in vitro model of IFNγ-induced persistence. PMID:24959423

A novel taspine derivative suppresses human liver tumor growth and invasion in vitro and in vivo.

PubMed

Wang, Nan; Zheng, Lei; Zhan, Yingzhuan; Zhang, Yanmin

2013-09-01

Taspine is an attractive target of research due to the anticancer and anti-angiogenic effects shown by in vitro and in vivo experiments. The present study investigated the role of tas1611, which is a derivative of taspine that has increased activity and solubility, in the regulation of the invasive properties of the SMMC-7721 liver cell line in vitro and in tumor inhibition in vivo . The proliferation of the SMMC-7721 cells was examined using the tetrazole blue colorimetric method. Matrigel ® invasion chamber assays and zymogram analyses were performed to assess the inhibitory effect of tas1611 on cell invasion. Finally, a solid tumor athymic mouse model was employed to further investigate the anti-tumor effect of this compound. The results revealed that tas1611 had a marked inhibitory effect on the invasion of the SMMC-7721 cells and that this effect was associated with the activity and expression levels of matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, tas1611 was able to inhibit tumor growth effectively in a solid tumor SMMC-7721 athymic mouse model. In conclusion, tas1611 may serve as a promising novel therapeutic candidate for the treatment of metastatic liver cancer.

The healing process of intracorporeally and in situ devitalized distal femur by microwave in a dog model and its mechanical properties in vitro.

PubMed

Ji, Zhenwei; Ma, Yunlei; Li, Wei; Li, Xiaoxiang; Zhao, Guangyi; Yun, Zhe; Qian, Jixian; Fan, Qingyu

2012-01-01

Limb-salvage surgery has been well recognized as a standard treatment and alternative to amputation for patients with malignant bone tumors. Various limb-sparing techniques have been developed including tumor prosthesis, allograft, autograft and graft-prosthesis composite. However, each of these methods has short- and long-term disadvantages such as nonunion, mechanical failures and poor limb function. The technique of intracorporeal devitalization of tumor-bearing bone segment in situ by microwave-induced hyperthermia after separating it from surrounding normal tissues with a safe margin is a promising limb-salvage method, which may avoid some shortcomings encountered by the above-mentioned conventional techniques. The purpose of this study is to assess the healing process and revitalization potential of the devitalized bone segment by this method in a dog model. In addition, the immediate effect of microwave on the biomechanical properties of bone tissue was also explored in an in vitro experiment. We applied the microwave-induced hyperthermia to devitalize the distal femurs of dogs in situ. Using a monopole microwave antenna, we could produce a necrotic bone of nearly 20 mm in length in distal femur. Radiography, bone scintigraphy, microangiography, histology and functional evaluation were performed at 2 weeks and 1, 2, 3, 6, 9 and 12 months postoperatively to assess the healing process. In a biomechanical study, two kinds of bone specimens, 3 and 6 cm in length, were used for compression and three-point bending test respectively immediately after extracorporeally devitalized by microwave. An in vivo study showed that intracorporeally and in situ devitalized bone segment by microwave had great revitalization potential. An in vitro study revealed that the initial mechanical strength of the extracorporeally devitalized bone specimen may not be affected by microwave. Our results suggest that the intracorporeal microwave devitalization of tumor-bearing bone segment in situ may be a promising limb-salvage method.

Effects of P-MAPA Immunomodulator on Toll-Like Receptors and p53: Potential Therapeutic Strategies for Infectious Diseases and Cancer

PubMed Central

2012-01-01

Background Compounds that can act as agonists for toll-like receptors (TLRs) may be promising candidates for the development of drugs against infectious diseases and cancer. The present study aimed to characterize the immunomodulatory effects of P-MAPA on TLRs in vitro and in vivo, as well as to investigate its potential as adjuvant therapy in infectious diseases and cancer. Methods For these purposes, the activity of P-MAPA on TLRs was assayed in vitro through NF-κB activation in HEK293 cells expressing a given TLR, and using an in vivo animal model for bladder cancer (BC). The antimicrobial activity of P-MAPA was tested against Mycobacterium tuberculosis (TB) in vitro in an MIC assay, and in vivo using an aerosol infection model of murine tuberculosis. Antitumor effects of P-MAPA were tested in an animal model with experimentally induced BC. Moxifloxacin (MXF) and Bacillus Calmette-Guerin (BCG) were used as positive controls in the animal models. Results The results showed that P-MAPA, administered alone or in combination with MXF, induced significant responses in vivo against TB. In contrast, the compound did not show antimicrobial activity in vitro. P-MAPA showed a significant stimulatory effect on human TLR2 and TLR4 in vitro. In BC, TLR2, TLR4 and p53 protein levels were significantly higher in the P-MAPA group than in the BCG group. The most common histopathological changes in each group were papillary carcinoma in BC group, low-grade intraepithelial neoplasia in BCG group and simple hyperplasia in P-MAPA group. Concerning the toxicological analysis performed during BC treatment, P-MAPA did not show evidence for hepatotoxicity and nephrotoxicity. Conclusions In conclusion, P-MAPA acted as TLR ligand in vitro and improved the immunological status in BC, increasing TLR2 and TLR4 protein levels. P-MAPA immunotherapy was more effective in restoring p53 and TLRs reactivities and showed significantly greater antitumor activity than BCG. The activation of TLRs and p53 may provide a hypothetical mechanism for the therapeutic effects in both cancer and infectious diseases. Taken together data obtained will encourage the further investigation of P-MAPA as a potential candidate for the treatment of cancer and infectious diseases. PMID:22709446

Effects of silver nanoparticles on the liver and hepatocytes in vitro.

PubMed

Gaiser, Birgit K; Hirn, Stephanie; Kermanizadeh, Ali; Kanase, Nilesh; Fytianos, Kleanthis; Wenk, Alexander; Haberl, Nadine; Brunelli, Andrea; Kreyling, Wolfgang G; Stone, Vicki

2013-02-01

With the increasing use and incorporation of nanoparticles (NPs) into consumer products, screening for potential toxicity is necessary to ensure customer safety. NPs have been shown to translocate to the bloodstream following inhalation and ingestion, and such studies demonstrate that the liver is an important organ for accumulation. Silver (Ag) NPs are highly relevant for human exposure due to their use in food contact materials, dietary supplements, and antibacterial wound treatments. Due to the large number of different NPs already used in various products and being developed for new applications, it is essential that relevant, quick, and cheap methods of in vitro risk assessment suitable for these new materials are established. Therefore, this study used a simple hepatocytes model combined with an in vivo injection model to simulate the passage of a small amount of NPs into the bloodstream following exposure, e.g., via ingestion or inhalation, and examined the potential of Ag NPs of 20 nm diameter to cause toxicity, inflammation, and oxidative stress in the liver following in vivo exposures of female Wistar rats via iv injection to 50 μg of NPs and in vitro exposures using the human hepatocyte cell line C3A. We found that Ag NPs were highly cytotoxic to hepatocytes (LC(50) lactate dehydrogenase: 2.5 μg/cm(2)) and affected hepatocyte homeostasis by reducing albumin release. At sublethal concentrations with normal cell or tissue morphology, Ag NPs were detected in cytoplasm and nuclei of hepatocytes. We observed similar effects of Ag NPs on inflammatory mediator expression in vitro and in vivo with increase of interleukin-8 (IL-8)/macrophage inflammatory protein 2, IL-1RI, and tumor necrosis factor-α expression in both models and increased IL-8 protein release in vitro. This article presents evidence of the potential toxicity and inflammogenic potential of Ag NPs in the liver following ingestion. In addition, the similarities between in vitro and in vivo responses are striking and encouraging for future reduction, refinement, and replacement of animal studies by the use of hepatocyte cell lines in particle risk assessment.

Temporary Vascular Occlusion by Rapid Reverse Phase Polymer: A Preliminary In Vitro Study of Retrograde Injection

PubMed Central

Dregelid, Einar

2012-01-01

During vascular surgical operations, there is a need for a simpler and more reliable method of temporary arterial occlusion than those currently employed, especially of heavily calcified arteries. A thermosensitive polymer, LeGoo (LG) (Pluromed, Woburn, MA), has been used successfully for temporary vascular occlusion. It has hitherto been injected by a cannula that has been introduced into the artery to be occluded, here henceforth called the “cannulation method.” Injection into arterial ostia without cannulation, using an injection device that arrests blood flow during the injection, here henceforth called “a retrograde method” may enable temporary hemostasis when ostial stenoses render it impossible to inject LG using the cannulation method. The objective of the present study was to study the feasibility of a retrograde method and to compare it with the cannulation method in an in vitro model, incorporating a narrow orifice to simulate ostial stenosis, using tap water at 37°C instead of blood. The retrograde method of LG injection, using a modified paediatric Foley catheter, turned out to be feasible to produce a durable LG plug more reliably, at higher water pressure and with less deep LG injection than with the cannulation method. PMID:22888352

Cheminformatics Analysis of EPA ToxCast Chemical Libraries ...

EPA Pesticide Factsheets

An important goal of toxicology research is the development of robust methods that use in vitro and chemical structure information to predict in vivo toxicity endpoints. The US EPA ToxCast program is addressing this goal using ~600 in vitro assays to create bioactivity profiles on a set of 320 compounds, mostly pesticide actives, that have well characterized in vivo toxicity. These 320 compounds (EPA-320 set evaluated in Phase I of ToxCast) are a subset of a much larger set of ~10,000 candidates that are of interest to the EPA (called here EPA-10K). Predictive models of in vivo toxicity are being constructed from the in vitro assay data on the EPA-320 chemical set. These models require validation on additional chemicals prior to wide acceptance, and this will be carried out by evaluating compounds from EPA-10K in Phase II of ToxCast. We have used cheminformatics approaches including clustering, data visualization, and QSAR to develop models for EPA-320 that could help prioritizing EPA-10K validation chemicals. Both chemical descriptors, as well as calculated physicochemical properties have been used. Compounds from EPA-10K are prioritized based on their similarity to EPA-320 using different similarity metrics, with similarity thresholds defining the domain of applicability for the predictive models built for EPA-320 set. In addition, prioritized lists of compounds of increasing dissimilarity from the EPA-320 have been produced, to test the ability of the EPA-320

Generation of stable PDX derived cell lines using conditional reprogramming.

PubMed

Borodovsky, Alexandra; McQuiston, Travis J; Stetson, Daniel; Ahmed, Ambar; Whitston, David; Zhang, Jingwen; Grondine, Michael; Lawson, Deborah; Challberg, Sharon S; Zinda, Michael; Pollok, Brian A; Dougherty, Brian A; D'Cruz, Celina M

2017-12-06

Efforts to develop effective cancer therapeutics have been hindered by a lack of clinically predictive preclinical models which recapitulate this complex disease. Patient derived xenograft (PDX) models have emerged as valuable tools for translational research but have several practical limitations including lack of sustained growth in vitro. In this study, we utilized Conditional Reprogramming (CR) cell technology- a novel cell culture system facilitating the generation of stable cultures from patient biopsies- to establish PDX-derived cell lines which maintain the characteristics of the parental PDX tumor. Human lung and ovarian PDX tumors were successfully propagated using CR technology to create stable explant cell lines (CR-PDX). These CR-PDX cell lines maintained parental driver mutations and allele frequency without clonal drift. Purified CR-PDX cell lines were amenable to high throughput chemosensitivity screening and in vitro genetic knockdown studies. Additionally, re-implanted CR-PDX cells proliferated to form tumors that retained the growth kinetics, histology, and drug responses of the parental PDX tumor. CR technology can be used to generate and expand stable cell lines from PDX tumors without compromising fundamental biological properties of the model. It offers the ability to expand PDX cells in vitro for subsequent 2D screening assays as well as for use in vivo to reduce variability, animal usage and study costs. The methods and data detailed here provide a platform to generate physiologically relevant and predictive preclinical models to enhance drug discovery efforts.

In vitro versus in vivo protein digestibility techniques for calculating PDCAAS (protein digestibility-corrected amino acid score) applied to chickpea fractions.

PubMed

Tavano, Olga Luisa; Neves, Valdir Augusto; da Silva Júnior, Sinézio Inácio

2016-11-01

Seven different in vitro methods to determine the protein digestibility for chickpea proteins were considered and also the application of these methodologies for calculating PDCAAS (protein digestibility-corrected amino acid score), seeking their correlations with the in vivo methodology. In vitro digestibility of raw and heated samples were determined using pepsin-pancreatin hydrolysis, considering soluble nitrogen via Kjeldahl (ppKJ) and hydrolysed peptide linkages using trinitrobenzenesulfonic acid and o-phthaldialdehyde. In vitro digestibility was also determined using trypsin, chymotrypsin and peptidase (3-Enz) or trypsin, chymotrypsin, peptidase and pronase solution (4-Enz). None of the correlations between in vitro and in vivo digestibilities were significant (at p<0.0500), but, strong correlations were observed between PDCAAS calculated by in vitro and in vivo results. PDCAAS-ppKJ, PDCAAS-3-Enz and PDCAAS-4-Enz presented the highest correlations with in vivo method, r=0.9316, 0.9442 and 0.9649 (p<0.0500), respectively. The use of in vitro methods for calculating PDCAAS may be promising and deserves more discussions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Toxicokinetic Triage for Environmental Chemicals.

PubMed

Wambaugh, John F; Wetmore, Barbara A; Pearce, Robert; Strope, Cory; Goldsmith, Rocky; Sluka, James P; Sedykh, Alexander; Tropsha, Alex; Bosgra, Sieto; Shah, Imran; Judson, Richard; Thomas, Russell S; Setzer, R Woodrow

2015-09-01

Toxicokinetic (TK) models link administered doses to plasma, blood, and tissue concentrations. High-throughput TK (HTTK) performs in vitro to in vivo extrapolation to predict TK from rapid in vitro measurements and chemical structure-based properties. A significant toxicological application of HTTK has been "reverse dosimetry," in which bioactive concentrations from in vitro screening studies are converted into in vivo doses (mg/kg BW/day). These doses are predicted to produce steady-state plasma concentrations that are equivalent to in vitro bioactive concentrations. In this study, we evaluate the impact of the approximations and assumptions necessary for reverse dosimetry and develop methods to determine whether HTTK tools are appropriate or may lead to false conclusions for a particular chemical. Based on literature in vivo data for 87 chemicals, we identified specific properties (eg, in vitro HTTK data, physico-chemical descriptors, and predicted transporter affinities) that correlate with poor HTTK predictive ability. For 271 chemicals we developed a generic HT physiologically based TK (HTPBTK) model that predicts non-steady-state chemical concentration time-courses for a variety of exposure scenarios. We used this HTPBTK model to find that assumptions previously used for reverse dosimetry are usually appropriate, except most notably for highly bioaccumulative compounds. For the thousands of man-made chemicals in the environment that currently have no TK data, we propose a 4-element framework for chemical TK triage that can group chemicals into 7 different categories associated with varying levels of confidence in HTTK predictions. For 349 chemicals with literature HTTK data, we differentiated those chemicals for which HTTK approaches are likely to be sufficient, from those that may require additional data. Published by Oxford University Press on behalf of Society of Toxicology 2015. This work is written by US Government employees and is in the public domain in the US.

Development and validation of in vitro-in vivo correlation (IVIVC) for estradiol transdermal drug delivery systems.

PubMed

Yang, Yang; Manda, Prashanth; Pavurala, Naresh; Khan, Mansoor A; Krishnaiah, Yellela S R

2015-07-28

The objective of this study was to develop a level A in vitro-in vivo correlation (IVIVC) for drug-in-adhesive (DIA) type estradiol transdermal drug delivery systems (TDDS). In vitro drug permeation studies across human skin were carried out to obtain the percent of estradiol permeation from marketed products. The in vivo time versus plasma concentration data of three estradiol TDDS at drug loadings of 2.0, 3.8 and 7.6mg (delivery rates of 25, 50 and 100μg/day, respectively) was deconvoluted using Wagner-Nelson method to obtain percent of in vivo drug absorption in postmenopausal women. The IVIVC between the in vitro percent of drug permeation (X) and in vivo percent of drug absorption (Y) for these three estradiol TDDS was constructed using GastroPlus® software. There was a high correlation (R(2)=1.0) with a polynomial regression of Y=-0.227X(2)+0.331X-0.001. These three estradiol TDDS were used for internal validation whereas another two products of the same formulation design (with delivery rates of 60 and 100μg/day) were used for external validation. The predicted estradiol serum concentrations (convoluted from in vitro skin permeation data) were compared with the observed serum concentrations for the respective products. The developed IVIVC model passed both the internal and external validations as the prediction errors (%PE) for Cmax and AUC were less than 15%. When another marketed estradiol TDDS with a delivery rate of 100μg/day but with a slight variation in formulation design was chosen, it did not pass external validation indicating the product-specific nature of IVIVC model. Results suggest that the IVIVC model developed in this study can be used to successfully predict the in vivo performance of the same estradiol TDDS with in vivo delivery rates ranging from 25 to 100μg/day. Published by Elsevier B.V.

Bayesian models trained with HTS data for predicting β-haematin inhibition and in vitro antimalarial activity.

PubMed

Wicht, Kathryn J; Combrinck, Jill M; Smith, Peter J; Egan, Timothy J

2015-08-15

A large quantity of high throughput screening (HTS) data for antimalarial activity has become available in recent years. This includes both phenotypic and target-based activity. Realising the maximum value of these data remains a challenge. In this respect, methods that allow such data to be used for virtual screening maximise efficiency and reduce costs. In this study both in vitro antimalarial activity and inhibitory data for β-haematin formation, largely obtained from publically available sources, has been used to develop Bayesian models for inhibitors of β-haematin formation and in vitro antimalarial activity. These models were used to screen two in silico compound libraries. In the first, the 1510 U.S. Food and Drug Administration approved drugs available on PubChem were ranked from highest to lowest Bayesian score based on a training set of β-haematin inhibiting compounds active against Plasmodium falciparum that did not include any of the clinical antimalarials or close analogues. The six known clinical antimalarials that inhibit β-haematin formation were ranked in the top 2.1% of compounds. Furthermore, the in vitro antimalarial hit-rate for this prioritised set of compounds was found to be 81% in the case of the subset where activity data are available in PubChem. In the second, a library of about 5000 commercially available compounds (Aldrich(CPR)) was virtually screened for ability to inhibit β-haematin formation and then for in vitro antimalarial activity. A selection of 34 compounds was purchased and tested, of which 24 were predicted to be β-haematin inhibitors. The hit rate for inhibition of β-haematin formation was found to be 25% and a third of these were active against P. falciparum, corresponding to enrichments estimated at about 25- and 140-fold relative to random screening, respectively. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterization of the biology and infectivity of Leishmania infantum viscerotropic and dermotropic strains isolated from HIV+ and HIV- patients in the murine model of visceral leishmaniasis

PubMed Central

2013-01-01

Background Leishmaniasis is a group of diseases with a variety of clinical manifestations. The form of the disease is highly dependent on the infective Leishmania species and the immunological status of the host. The infectivity of the parasite strain also plays an important role in the progression of the infection. The aim of this work is to understand the influence of the natural infectivity of Leishmania strains in the outcome of visceral leishmaniasis. Methods In this study we have characterized four strains of L. infantum in terms of molecular typing, in vitro cultivation and differentiation. Two strains were isolated from HIV+ patients with visceral leishmaniasis (Bibiano and E390M), one strain was isolated from a cutaneous lesion in an immunocompetent patient (HL) and another internal reference strain causative of visceral leishmaniasis (ST) also from an immunocompetent patient was used for comparison. For this objective, we have compared their virulence by in vitro and in vivo infectivity in a murine model of visceral leishmaniasis. Results Molecular typing unraveled a new k26 sequence attributed to MON-284 zymodeme and allowed the generation of a molecular signature for the identification of each strain. In vitro cultivation enabled the production of promastigotes with comparable growth curves and metacyclogenesis development. The HL strain was the most infective, showing the highest parasite loads in vitro that were corroborated with the in vivo assays, 6 weeks post-infection in BALB/c mice. The two strains isolated from HIV+ patients, both belonging to two different zymodemes, revealed different kinetics of infection. Conclusion Differences in in vitro and in vivo infectivity found in the murine model were then attributed to intrinsic characteristics of each strain. This work is supported by other studies that present the parasite’s inherent features as factors for the multiplicity of clinical manifestations and severity of leishmaniasis. PMID:23622683

76 FR 29752 - Nomination of In Vitro Test Methods for Detection and Quantification of Botulinum Neurotoxins and...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-05-23

... DEPARTMENT OF HEALTH AND HUMAN SERVICES Nomination of In Vitro Test Methods for Detection and... Evaluated by These Test Methods AGENCY: Division of National Toxicology Program (NTP), National Institute of... Methods (ICCVAM), the NTP Interagency Center for the Evaluation of Alternative Toxicological Methods...

The proof-of-concept of ASS234: Peripherally administered ASS234 enters the central nervous system and reduces pathology in a male mouse model of Alzheimer disease

PubMed Central

Serrano, Mari Paz; Herrero-Labrador, Raquel; Futch, Hunter S.; Serrano, Julia; Romero, Alejandro; Fernandez, Ana Patricia; Samadi, Abdelouahid; Unzeta, Mercedes; Marco-Contelles, Jose; Martínez-Murillo, Ricardo

2017-01-01

Background The heterogeneity of Alzheimer disease requires the development of multitarget drugs for treating the symptoms of the disease and its progression. Both cholinergic and monoamine oxidase dysfunctions are involved in the pathological process. Thus, we hypothesized that the development of therapies focused on these targets might be effective. We have developed and assessed a new product, coded ASS234, a multipotent acetyl and butyrylcholinesterase/monoamine oxidase A–B inhibitor with a potent inhibitory effect on amyloid-β aggregation as well as antioxidant and antiapoptotic properties. But there is a need to reliably correlate in vitro and in vivo drug release data. Methods We examined the effect of ASS234 on cognition in healthy adult C57BL/6J mice in a model of scopolamine-induced cognitive impairment that often accompanies normal and pathological aging. Also, in a characterized transgenic APPswe/PS1ΔE9 mouse model of Alzheimer disease, we examined the effects of short-term ASS234 treatment on plaque deposition and gliosis using immunohistochemistry. Toxicology of ASS234 was assessed using a quantitative high-throughput in vitro cytotoxicity screening assay following the MTT assay method in HepG2 liver cells. Results In vivo, ASS234 significantly decreased scopolamine-induced learning deficits in C57BL/6J mice. Also, reduction of amyloid plaque burden and gliosis in the cortex and hippocampus was assessed. In vitro, ASS234 exhibited lesser toxicity than donepezil and tacrine. Limitations The study was conducted in male mice only. Although the Alzheimer disease model does not recapitulate all features of the human disease, it exhibits progressive monoaminergic neurodegeneration. Conclusion ASS234 is a promising alternative drug of choice to treat the cognitive decline and neurodegeneration underlying Alzheimer disease. PMID:27636528

A quantitative framework to evaluate modeling of cortical development by neural stem cells

PubMed Central

Stein, Jason L.; de la Torre-Ubieta, Luis; Tian, Yuan; Parikshak, Neelroop N.; Hernandez, Israel A.; Marchetto, Maria C.; Baker, Dylan K.; Lu, Daning; Hinman, Cassidy R.; Lowe, Jennifer K.; Wexler, Eric M.; Muotri, Alysson R.; Gage, Fred H.; Kosik, Kenneth S.; Geschwind, Daniel H.

2014-01-01

Summary Neural stem cells have been adopted to model a wide range of neuropsychiatric conditions in vitro. However, how well such models correspond to in vivo brain has not been evaluated in an unbiased, comprehensive manner. We used transcriptomic analyses to compare in vitro systems to developing human fetal brain and observed strong conservation of in vivo gene expression and network architecture in differentiating primary human neural progenitor cells (phNPCs). Conserved modules are enriched in genes associated with ASD, supporting the utility of phNPCs for studying neuropsychiatric disease. We also developed and validated a machine learning approach called CoNTExT that identifies the developmental maturity and regional identity of in vitro models. We observed strong differences between in vitro models, including hiPSC-derived neural progenitors from multiple laboratories. This work provides a systems biology framework for evaluating in vitro systems and supports their value in studying the molecular mechanisms of human neurodevelopmental disease. PMID:24991955

RNA sequencing analysis reveals quiescent microglia isolation methods from postnatal mouse brains and limitations of BV2 cells.

PubMed

He, Yingbo; Yao, Xiang; Taylor, Natalie; Bai, Yuchen; Lovenberg, Timothy; Bhattacharya, Anindya

2018-05-22

Microglia play key roles in neuron-glia interaction, neuroinflammation, neural repair, and neurotoxicity. Currently, various microglial in vitro models including primary microglia derived from distinct isolation methods and immortalized microglial cell lines are extensively used. However, the diversity of these existing models raises difficulty in parallel comparison across studies since microglia are sensitive to environmental changes, and thus, different models are likely to show widely varied responses to the same stimuli. To better understand the involvement of microglia in pathophysiological situations, it is critical to establish a reliable microglial model system. With postnatal mouse brains, we isolated microglia using three general methods including shaking, mild trypsinization, and CD11b magnetic-associated cell sorting (MACS) and applied RNA sequencing to compare transcriptomes of the isolated cells. Additionally, we generated a genome-wide dataset by RNA sequencing of immortalized BV2 microglial cell line to compare with primary microglia. Furthermore, based on the outcomes of transcriptional analysis, we compared cellular functions between primary microglia and BV2 cells including immune responses to LPS by quantitative RT-PCR and Luminex Multiplex Assay, TGFβ signaling probed by Western blot, and direct migration by chemotaxis assay. We found that although the yield and purity of microglia were comparable among the three isolation methods, mild trypsinization drove microglia in a relatively active state, evidenced by high amount of amoeboid microglia, enhanced expression of microglial activation genes, and suppression of microglial quiescent genes. In contrast, CD11b MACS was the most reliable and consistent method, and microglia isolated by this method maintained a relatively resting state. Transcriptional and functional analyses revealed that as compared to primary microglia, BV2 cells remain most of the immune functions such as responses to LPS but showed limited TGFβ signaling and chemotaxis upon chemoattractant C5a. Collectively, we determined the optimal isolation methods for quiescent microglia and characterized the limitations of BV2 cells as an alternative of primary microglia. Considering transcriptional and functional differences, caution should be taken when extrapolating data from various microglial models. In addition, our RNA sequencing database serves as a valuable resource to provide novel insights for appropriate application of microglia as in vitro models.

RAT PLACENTATION: AN EXPERIMENTAL MODEL FOR INVESTIGATING THE HEMOCHORIAL MATERNAL-FETAL INTERFACE

PubMed Central

Soares, Michael J.; Chakraborty, Damayanti; Rumi, M.A. Karim; Konno, Toshihiro; Renaud, Stephen J.

2011-01-01

The rat possesses hemochorial placentation with deep intrauterine trophoblast cell invasion and trophoblast-directed uterine spiral artery remodeling; features shared with human placentation. Recognition of these similarities spurred the establishment of in vitro and in vivo research methods using the rat as an animal model to address mechanistic questions regarding development of the hemochorial placenta. The purpose of this review is to provide the requisite background to help move the rat to the forefront in placentation research. PMID:22284666

A coupling method for a cardiovascular simulation model which includes the Kalman filter.

PubMed

Hasegawa, Yuki; Shimayoshi, Takao; Amano, Akira; Matsuda, Tetsuya

2012-01-01

Multi-scale models of the cardiovascular system provide new insight that was unavailable with in vivo and in vitro experiments. For the cardiovascular system, multi-scale simulations provide a valuable perspective in analyzing the interaction of three phenomenons occurring at different spatial scales: circulatory hemodynamics, ventricular structural dynamics, and myocardial excitation-contraction. In order to simulate these interactions, multiscale cardiovascular simulation systems couple models that simulate different phenomena. However, coupling methods require a significant amount of calculation, since a system of non-linear equations must be solved for each timestep. Therefore, we proposed a coupling method which decreases the amount of calculation by using the Kalman filter. In our method, the Kalman filter calculates approximations for the solution to the system of non-linear equations at each timestep. The approximations are then used as initial values for solving the system of non-linear equations. The proposed method decreases the number of iterations required by 94.0% compared to the conventional strong coupling method. When compared with a smoothing spline predictor, the proposed method required 49.4% fewer iterations.

Extended Latanoprost Release from Commercial Contact Lenses: In Vitro Studies Using Corneal Models

PubMed Central

Mohammadi, Saman; Jones, Lyndon; Gorbet, Maud

2014-01-01

In this study, we compared, for the first time, the release of a 432 kDa prostaglandin analogue drug, Latanoprost, from commercially available contact lenses using in vitro models with corneal epithelial cells. Conventional polyHEMA-based and silicone hydrogel soft contact lenses were soaked in drug solution ( solution in phosphate buffered saline). The drug release from the contact lens material and its diffusion through three in vitro models was studied. The three in vitro models consisted of a polyethylene terephthalate (PET) membrane without corneal epithelial cells, a PET membrane with a monolayer of human corneal epithelial cells (HCEC), and a PET membrane with stratified HCEC. In the cell-based in vitro corneal epithelium models, a zero order release was obtained with the silicone hydrogel materials (linear for the duration of the experiment) whereby, after 48 hours, between 4 to 6 of latanoprost (an amount well within the range of the prescribed daily dose for glaucoma patients) was released. In the absence of cells, a significantly lower amount of drug, between 0.3 to 0.5 , was released, (). The difference observed in release from the hydrogel lens materials in the presence and absence of cells emphasizes the importance of using an in vitro corneal model that is more representative of the physiological conditions in the eye to more adequately characterize ophthalmic drug delivery materials. Our results demonstrate how in vitro models with corneal epithelial cells may allow better prediction of in vivo release. It also highlights the potential of drug-soaked silicone hydrogel contact lens materials for drug delivery purposes. PMID:25207851

Fatty acids rather than hormones restore in vitro angiogenesis in human male and female endothelial cells cultured in charcoal-stripped serum

PubMed Central

Vanetti, Claudia; Bifari, Francesco; Vicentini, Lucia M.

2017-01-01

Charcoal-stripped serum (CSS) is a well-accepted method to model effects of sex hormones in cell cultures. We have recently shown that human endothelial cells (ECs) fail to growth and to undergo in vitro angiogenesis when cultured in CSS. However, the mechanism(s) underlying the CSS-induced impairment of in vitro EC properties are still unknown. In addition, whether there is any sexual dimorphism in the CSS-induced EC phenotype remains to be determined. Here, by independently studying human male and female ECs, we found that CSS inhibited both male and female EC growth and in vitro angiogenesis, with a more pronounced effect on male EC sprouting. Reconstitution of CSS with 17-β estradiol, dihydrotestosterone, or the lipophilic thyroid hormone did not restore EC functions in both sexes. On the contrary, supplementation with palmitic acid or the acetyl-CoA precursor acetate significantly rescued the CSS-induced inhibition of growth and sprouting in both male and female ECs. We can conclude that the loss of metabolic precursors (e.g., fatty acids) rather than of hormones is involved in the impairment of in vitro proliferative and angiogenic properties of male and female ECs cultured with CSS. PMID:29232396

Imatinib prevents beta cell death in vitro but does not improve islet transplantation outcome

PubMed Central

Griffiths, Lisa A.; Persaud, Shanta J.; Jones, Peter M.; Howell, Simon L.; Welsh, Nils

2016-01-01

Introduction Improving islet transplantation outcome could not only bring benefits to individual patients but also widen the patient pool to which this life-changing treatment is available. Imatinib has previously been shown to protect beta cells from apoptosis in a variety of in vitro and in vivo models. The aim of this study was to investigate whether imatinib could be used to improve islet transplantation outcome. Methods Islets were isolated from C57Bl/6 mice and pre-cultured with imatinib prior to exposure to streptozotocin and cytokines in vitro. Cell viability and glucose-induced insulin secretion were measured. For transplantation experiments, islets were pre-cultured with imatinib for either 72 h or 24 h prior to transplantation into streptozotocin-diabetic C57Bl/6 mice. In one experimental series mice were also administered imatinib after islet transplantation. Results Imatinib partially protected islets from beta cell death in vitro. However, pre-culturing islets in imatinib or administering the drug to the mice in the days following islet transplantation did not improve blood glucose concentrations more than control-cultured islets. Conclusion Although imatinib protected against beta cell death from cytokines and streptozotocin in vitro, it did not significantly improve syngeneic islet transplantation outcome. PMID:26953716

Imatinib prevents beta cell death in vitro but does not improve islet transplantation outcome.

PubMed

King, Aileen J F; Griffiths, Lisa A; Persaud, Shanta J; Jones, Peter M; Howell, Simon L; Welsh, Nils

2016-05-01

Introduction Improving islet transplantation outcome could not only bring benefits to individual patients but also widen the patient pool to which this life-changing treatment is available. Imatinib has previously been shown to protect beta cells from apoptosis in a variety of in vitro and in vivo models. The aim of this study was to investigate whether imatinib could be used to improve islet transplantation outcome. Methods Islets were isolated from C57Bl/6 mice and pre-cultured with imatinib prior to exposure to streptozotocin and cytokines in vitro. Cell viability and glucose-induced insulin secretion were measured. For transplantation experiments, islets were pre-cultured with imatinib for either 72 h or 24 h prior to transplantation into streptozotocin-diabetic C57Bl/6 mice. In one experimental series mice were also administered imatinib after islet transplantation. Results Imatinib partially protected islets from beta cell death in vitro. However, pre-culturing islets in imatinib or administering the drug to the mice in the days following islet transplantation did not improve blood glucose concentrations more than control-cultured islets. Conclusion Although imatinib protected against beta cell death from cytokines and streptozotocin in vitro, it did not significantly improve syngeneic islet transplantation outcome.

Advances in In Vitro and In Silico Tools for Toxicokinetic Dose ...

EPA Pesticide Factsheets

Recent advances in vitro assays, in silico tools, and systems biology approaches provide opportunities for refined mechanistic understanding for chemical safety assessment that will ultimately lead to reduced reliance on animal-based methods. With the U.S. commercial chemical landscape encompassing thousands of chemicals with limited data, safety assessment strategies that reliably predict in vivo systemic exposures and subsequent in vivo effects efficiently are a priority. Quantitative in vitro-in vivo extrapolation (QIVIVE) is a methodology that facilitates the explicit and quantitative application of in vitro experimental data and in silico modeling to predict in vivo system behaviors and can be applied to predict chemical toxicokinetics, toxicodynamics and also population variability. Tiered strategies that incorporate sufficient information to reliably inform the relevant decision context will facilitate acceptance of these alternative data streams for safety assessments. This abstract does not necessarily reflect U.S. EPA policy. This talk will provide an update to an international audience on the state of science being conducted within the EPA’s Office of Research and Development to develop and refine approaches that estimate internal chemical concentrations following a given exposure, known as toxicokinetics. Toxicokinetic approaches hold great potential in their ability to link in vitro activities or toxicities identified during high-throughput screen

Pulse-wave propagation in straight-geometry vessels for stiffness estimation: theory, simulations, phantoms and in vitro findings.

PubMed

Shahmirzadi, Danial; Li, Ronny X; Konofagou, Elisa E

2012-11-01

Pulse wave imaging (PWI) is an ultrasound-based method for noninvasive characterization of arterial stiffness based on pulse wave propagation. Reliable numerical models of pulse wave propagation in normal and pathological aortas could serve as powerful tools for local pulse wave analysis and a guideline for PWI measurements in vivo. The objectives of this paper are to (1) apply a fluid-structure interaction (FSI) simulation of a straight-geometry aorta to confirm the Moens-Korteweg relationship between the pulse wave velocity (PWV) and the wall modulus, and (2) validate the simulation findings against phantom and in vitro results. PWI depicted and tracked the pulse wave propagation along the abdominal wall of canine aorta in vitro in sequential Radio-Frequency (RF) ultrasound frames and estimates the PWV in the imaged wall. The same system was also used to image multiple polyacrylamide phantoms, mimicking the canine measurements as well as modeling softer and stiffer walls. Finally, the model parameters from the canine and phantom studies were used to perform 3D two-way coupled FSI simulations of pulse wave propagation and estimate the PWV. The simulation results were found to correlate well with the corresponding Moens-Korteweg equation. A high linear correlation was also established between PWV² and E measurements using the combined simulation and experimental findings (R² =  0.98) confirming the relationship established by the aforementioned equation.

Antimicrobial activity of root canal irrigants against biofilm forming pathogens- An in vitro study

PubMed Central

Ghivari, Sheetal Basavraj; Bhattacharya, Haimanti; Bhat, Kishore G.; Pujar, Madhu A.

2017-01-01

Aims: The aim of the study was to check the antimicrobial activity of the 5% Sodium hypochlorite, 2% Chlorhexidine, 0.10% Octenidine (OCT), and 2% Silver Zeolite (SZ) at different time intervals against a single species biofilm of Enterococcus faecalis, Staphylococcus aureus, and Candida albicans model prepared on a nitrocellulose membrane. Settings and Design: In vitro nitrocellulose biofilm model was used to check antibacterial efficacy of root canal irrigants. Materials and Methods: The in vitro nitrocellulose biofilm model was used to check the antibacterial activity of root canal irrigants. Single species biofilms were suspended into 96-well microtiter plate and treated with root canal irrigants for 1, 5, 10, 15, 30, and 60 s, respectively. The remaining microbial load in the form of colony-forming unit/ml after antimicrobial treatment was tabulated and data were statistically analyzed. Statistical Analysis: SPSS version 17, Kruskal–Wallis ANOVA, Mann–Whitney U-test, and Wilcoxon matched pair test (P < 0.05) were used. Results: All tested microorganisms were eliminated within 30 s by all the antimicrobial substances tested except normal saline. 2% chlorhexidine and 0.10% OCT were equally effective against C. albicans at 30 s. Conclusion: The newly tested irrigants have shown considerable antibacterial activity against selected single species biofilm. OCT (0.10%) can be used as an alternative endodontic irrigant. PMID:29279615

Establishment and Characterization of a New Muscle Cell Line of Zebrafish (Danio rerio) as an In Vitro Model for Gene Expression Studies.

PubMed

Kumar, Amit; Singh, Neha; Goswami, Mukunda; Srivastava, J K; Mishra, Akhilesh K; Lakra, W S

2016-01-01

A new continuous fibroblast cell line was established from the muscle tissue of healthy juvenile Danio rerio (Zebrafish) through explant method. Fish cell lines serve as useful tool for investigating basic fish biology, as a model for bioassay of environmental toxicant, toxicity ranking, and for developing molecular biomarkers. The cell line was continuously subcultured for a period of 12 months (61 passages) and maintained at 28 °C in L-15 medium supplemented with 10% FBS and 10 ng/mL of basic fibroblastic growth factor (bFGF) without use of antibiotics. Its growth rate was proportional to the FBS concentration, with optimum growth at 15% FBS. DNA barcoding (16SrRNA and COX1) was used to authenticate the cell line. Cells were incubated with propidium iodide and sorted via flow cytometry to calculate the DNA content to confirm the genetic stability. Significant green fluorescent protein (GFP) signals confirmed the utility of cell line in transgenic and genetic manipulation studies. In vitro assay was performed with MTT to examine the growth potential of the cell line. The muscle cell line would provide a novel invaluable in vitro model to identify important genes to understand regulatory mechanisms that govern the molecular regulation of myogenesis and should be useful in biomedical research.

Integrated Model of Chemical Perturbations of a Biological ...

EPA Pesticide Factsheets

We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation. The network model uses activity patterns across the in vitro assays to predict whether a chemical is an ER agonist or antagonist, or is otherwise influencing the assays through a manner dependent on the physics and chemistry of the technology platform (“”assay interference”). The method is applied to a library of 1812 commercial and environmental chemicals, including 45 ER positive and negative reference chemicals. Among the reference chemicals, the network model correctly identified the agonists and antagonists with the exception of very weak compounds whose activity was outside the concentration range tested. The model agonist score also correlated with the expected potency class of the active reference chemicals. Of the 1812 chemicals evaluated, 52 (2.8%) were predicted to be strongly ER active in agonist or antagonist mode. This dataset and model were also used to begin a systematic investigation of assay interference. The most prominent cause of false-positive activity (activity in an assay that is likely not due to interaction of the chemical with ER) is cytotoxicity. The model provides the ability to prioritize a large set of important environmental chemicals with human exposure potential for additional in v

Generation of a Close-to-Native In Vitro System to Study Lung Cells-Extracellular Matrix Crosstalk.

PubMed

Garlíková, Zuzana; Silva, Ana Catarina; Rabata, Anas; Potěšil, David; Ihnatová, Ivana; Dumková, Jana; Koledová, Zuzana; Zdráhal, Zbyněk; Vinarský, Vladimír; Hampl, Aleš; Pinto-do-Ó, Perpétua; Nascimento, Diana Santos

2018-01-01

Extracellular matrix (ECM) is an essential component of the tissue microenvironment, actively shaping cellular behavior. In vitro culture systems are often poor in ECM constituents, thus not allowing for naturally occurring cell-ECM interactions. This study reports on a straightforward and efficient method for the generation of ECM scaffolds from lung tissue and its subsequent in vitro application using primary lung cells. Mouse lung tissue was subjected to decellularization with 0.2% sodium dodecyl sulfate, hypotonic solutions, and DNase. Resultant ECM scaffolds were devoid of cells and DNA, whereas lung ECM architecture of alveolar region and blood and airway networks were preserved. Scaffolds were predominantly composed of core ECM and ECM-associated proteins such as collagens I-IV, nephronectin, heparan sulfate proteoglycan core protein, and lysyl oxidase homolog 1, among others. When homogenized and applied as coating substrate, ECM supported the attachment of lung fibroblasts (LFs) in a dose-dependent manner. After ECM characterization and biocompatibility tests, a novel in vitro platform for three-dimensional (3D) matrix repopulation that permits live imaging of cell-ECM interactions was established. Using this system, LFs colonized the ECM scaffolds, displaying a close-to-native morphology in intimate interaction with the ECM fibers, and showed nuclear translocation of the mechanosensor yes-associated protein (YAP), when compared with cells cultured in two dimensions. In conclusion, we developed a 3D-like culture system, by combining an efficient decellularization method with a live-imaging culture platform, to replicate in vitro native lung cell-ECM crosstalk. This is a valuable system that can be easily applied to other organs for ECM-related drug screening, disease modeling, and basic mechanistic studies.

Methods in Molecular Biology: Germline Stem Cells | Center for Cancer Research

Cancer.gov

The protocols in Germline Stem Cells are intended to present selected genetic, molecular, and cellular techniques used in germline stem cell research. The book is divided into two parts. Part I covers germline stem cell identification and regulation in model organisms. Part II covers current techniques used in in vitro culture and applications of germline stem cells.

20170308 - Higher Throughput Toxicokinetics to Allow ...

EPA Pesticide Factsheets

As part of "Ongoing EDSP Directions & Activities" I will present CSS research on high throughput toxicokinetics, including in vitro data and models to allow rapid determination of the real world doses that may cause endocrine disruption. This is a presentation as part of the U.S. Environmental Protection Agency – Japan Ministry of the Environment 12th Bilateral Meeting on Endocrine Disruption Test Methods Development.

An improved biofunction of titanium for keratoprosthesis by hydroxyapatite-coating.

PubMed

Dong, Ying; Yang, Jingxin; Wang, Liqiang; Ma, Xiao; Huang, Yifei; Qiu, Zhiye; Cui, Fuzhai

2014-03-01

Titanium framework keratoprosthesis has been commonly used in the severe corneal blindness, but the tissue melting occurred frequently around titanium. Since hydroxyapatite has been approved to possess a good tissue integration characteristic, nanostructured hydroxyapatite was coated on the surface of titanium through the aerosol deposition method. In this study, nanostructured hydroxyapatite coating was characterized by X-ray diffraction, scanning electron microscopy, atomic force microscopy, and auger electronic spectrometer. Biological evaluations were performed with rabbit cornea fibroblast in vitro and an animal model in vivo. The outcomes showed the coating had a grain-like surface topography and a good atomic mixed area with substrate. The rabbit cornea fibroblasts appeared a good adhesion on the surface of nanostructured hydroxyapatite in vitro. In the animal model, nanostructured hydroxyapatite-titanium implants were stably retained in the rabbit cornea, and by contrast, the corneal stroma became thinner anterior to the implants in the control. Therefore, our findings proved that nanostructured hydroxyapatite-titanium could not only provide an improved bond for substrate but also enhance the tissue integration with implants in host. As a promising material, nanostructured hydroxyapatite-titanium-based keratoprosthesis prepared by the aerosol deposition method could be utilized for the corneal blindness treatment.

From impedance theory to needle electrode guidance in tissue

NASA Astrophysics Data System (ADS)

Kalvøy, Håvard; Høyum, Per; Grimnes, Sverre; Martinsen, Ørjan G.

2010-04-01

Fast access to blood vessels or other tissues/organs can be crucial in clinical or acute medical treatment. We have developed a method for needle guidance for use in different types of applications. The feasibility of an automatic application for fast access to blood vessels during acute cardiac arrest, based on this method, has been evaluated. Suited electrode setups were found by development of needle electrode models used in simulation and sensitivity analyses. In vitro measurements were done both to determine the fundamental properties of the electrodes for use in the models and to confirm the simulation results. Development of algorithms for tissue characterization and differentiation was based on in vivo impedance measurement in porcine models and confirmed in human tissue in vivo. Feasibility was proven by application prototyping and impedance data presented as invasive Electrical Impedance Tomography (iEIT). Our conclusion is that this method can be utilized in a wide range of clinical applications.

In Vitro Determination of Drug Transfer from Drug-Coated Balloons

PubMed Central

Seidlitz, Anne; Kotzan, Nadine; Nagel, Stefan; Reske, Thomas; Grabow, Niels; Harder, Claus; Petersen, Svea; Sternberg, Katrin; Weitschies, Werner

2013-01-01

Drug-coated balloons are medical devices designed to locally deliver drug to diseased segments of the vessel wall. For these dosage forms, drug transfer to the vessel wall needs to be examined in detail, since drug released into the blood is cleared from the site. In order to examine drug transfer, a new in vitro setup was developed combining the estimation of drug loss during advancement to the site of application in a model coronary artery pathway with a hydrogel compartment representing, as a very simplified model, the vessel wall. The transfer of fluorescent model substances as well as the drug paclitaxel from coated balloons to the simulated vessel wall was evaluated using this method. The model was suitable to quantify the fractions transferred to the hydrogel and also to qualitatively assess distribution patterns in the hydrogel film. In the case of fluorescein sodium, rhodamin b and paclitaxel, vast amounts of the coated substance were lost during the simulated passage and only very small fractions of about 1% of the total load were transferred to the gel. This must be attributed to good water solubility of the fluorescent substances and the mechanical instability of the paclitaxel coating. Transfer of the hydrophobic model substance triamterene was however nearly unaffected by the preliminary tracking procedure with transferred fractions ranging from 8% to 14%. Analysis of model substance distribution yielded inhomogeneous distributions indicating that the coating was not evenly distributed on the balloon surface and that a great fraction of the coating liquid did not penetrate the folds of the balloon. This finding is contradictory to the generally accepted assumption of a drug depot inside the folds and emphasizes the necessity to thoroughly characterize in vitro performance of drug-coated balloons to support the very promising clinical data. PMID:24391863

Marginal and internal fit of cobalt-chromium copings fabricated using the conventional and the direct metal laser sintering techniques: A comparative in vitro study.

PubMed

Ullattuthodi, Sujana; Cherian, Kandathil Phillip; Anandkumar, R; Nambiar, M Sreedevi

2017-01-01

This in vitro study seeks to evaluate and compare the marginal and internal fit of cobalt-chromium copings fabricated using the conventional and direct metal laser sintering (DMLS) techniques. A master model of a prepared molar tooth was made using cobalt-chromium alloy. Silicone impression of the master model was made and thirty standardized working models were then produced; twenty working models for conventional lost-wax technique and ten working models for DMLS technique. A total of twenty metal copings were fabricated using two different production techniques: conventional lost-wax method and DMLS; ten samples in each group. The conventional and DMLS copings were cemented to the working models using glass ionomer cement. Marginal gap of the copings were measured at predetermined four points. The die with the cemented copings are standardized-sectioned with a heavy duty lathe. Then, each sectioned samples were analyzed for the internal gap between the die and the metal coping using a metallurgical microscope. Digital photographs were taken at ×50 magnification and analyzed using measurement software. Statistical analysis was done by unpaired t -test and analysis of variance (ANOVA). The results of this study reveal that no significant difference was present in the marginal gap of conventional and DMLS copings ( P > 0.05) by means of ANOVA. The mean values of internal gap of DMLS copings were significantly greater than that of conventional copings ( P < 0.05). Within the limitations of this in vitro study, it was concluded that the internal fit of conventional copings was superior to that of the DMLS copings. Marginal fit of the copings fabricated by two different techniques had no significant difference.

Evaluation of an alcohol-based surgical hand disinfectant containing a synergistic combination of farnesol and benzethonium chloride for immediate and persistent activity against resident hand flora of volunteers and with a novel in vitro pig skin model.

PubMed

Shintre, Milind S; Gaonkar, Trupti A; Modak, Shanta M

2007-02-01

To evaluate the immediate, persistent and sustained in vivo activity of an alcohol-based surgical hand disinfectant, consisting of a zinc gel and a preservative system containing a synergistic combination of farnesol and benzethonium chloride (ZBF disinfectant), and to develop a pig skin model for in vitro evaluation of the immediate and persistent efficacy of alcohol-based surgical hand disinfectants against resident hand flora. The in vivo immediate, persistent, and sustained activity of ZBF disinfectant was evaluated using human volunteers and the "glove-juice" method described in the US Food and Drug Administration's Tentative Final Monograph (FDA-TFM) for Healthcare Antiseptic Products. A novel in vitro pig skin model was developed to compare the immediate and persistent activity of alcohol-based surgical hand disinfectants against resident flora using Staphylococcus epidermidis as the test organism. Four alcohol-based surgical hand disinfectants were evaluated using this model. The results for the ZBF disinfectant exceed the FDA-TFM criteria for immediate, persistent, and sustained activity required for surgical hand disinfectants. The reduction factors for the 4 hand disinfectants obtained using the pig skin model show good agreement with the log(10) reductions in concentrations of hand flora obtained using human volunteers to test for immediate and persistent activity. The ZBF disinfectant we evaluated met the FDA-TFM criteria for surgical hand disinfectants. The immediate and persistent efficacy of the surgical hand disinfectants evaluated with the novel pig skin model described in this study shows good agreement with the results obtained in vivo.

Development of Ciprofloxacin-loaded contact lenses using fluorous chemistry

PubMed Central

Zhu, Zhiling; Li, Siheng; McDermott, Alison M.

2017-01-01

In this work, we developed a simple method to load drugs into commercially available contact lenses utilizing fluorous chemistry. We demonstrated this method using model compounds including fluorous-tagged fluorescein and antibiotic ciprofloxacin. We showed that fluorous interactions facilitated the loading of model molecules into fluorocarbon-containing contact lenses, and that the release profiles exhibited sustained release. Contact lenses loaded with fluorous-tagged ciprofloxacin exhibited antimicrobial activity against Pseudomonas aeruginosa in vitro, while no cytotoxicity towards human corneal epithelial cells was observed. To mimic the tear turnover, we designed a porcine eye infection model under flow conditions. Significantly, the modified lenses also exhibited antimicrobial efficacy against Pseudomonas aeruginosa in the ex vivo infection model. Overall, utilizing fluorous chemistry, we can construct a drug delivery system that exhibits high drug loading capacity, sustained drug release, and robust biological activity. PMID:28188995

Oral Administration of Ginseng Ameliorates Cyclosporine-Induced Pancreatic Injury in an Experimental Mouse Model

PubMed Central

Lim, Sun Woo; Doh, Kyoung Chan; Jin, Long; Piao, Shang Guo; Heo, Seong Beom; Zheng, Yu Fen; Bae, Soo Kyung; Chung, Byung Ha; Yang, Chul Woo

2013-01-01

Background This study was performed to investigate whether ginseng has a protective effect in an experimental mouse model of cyclosporine-induced pancreatic injury. Methods Mice were treated with cyclosporine (30 mg/kg/day, subcutaneously) and Korean red ginseng extract (0.2 or 0.4 g/kg/day, oral gavage) for 4 weeks while on a 0.01% salt diet. The effect of ginseng on cyclosporine-induced pancreatic islet dysfunction was investigated by an intraperitoneal glucose tolerance test and measurements of serum insulin level, β cell area, macrophage infiltration, and apoptosis. Using an in vitro model, we further examined the effect of ginseng on a cyclosporine-treated insulin-secreting cell line. Oxidative stress was measured by the concentration of 8-hydroxy-2′-deoxyguanosine in serum, tissue sections, and culture media. Results Four weeks of cyclosporine treatment increased blood glucose levels and decreased insulin levels, but cotreatment with ginseng ameliorated the cyclosporine-induced glucose intolerance and hyperglycemia. Pancreatic β cell area was also greater with ginseng cotreatment compared with cyclosporine monotherapy. The production of proinflammatory molecules, such as induced nitric oxide synthase and cytokines, and the level of apoptotic cell death also decreased in pancreatic β cell with ginseng treatment. Consistent with the in vivo results, the in vitro study showed that the addition of ginseng protected against cyclosporine-induced cytotoxicity, inflammation, and apoptotic cell death. These in vivo and in vitro changes were accompanied by decreases in the levels of 8-hydroxy-2′-deoxyguanosine in pancreatic β cell in tissue section, serum, and culture media during cotreatment of ginseng with cyclosporine. Conclusions The results of our in vivo and in vitro studies demonstrate that ginseng has a protective effect against cyclosporine-induced pancreatic β cell injury via reducing oxidative stress. PMID:24009697

Prescreening of Nicotine Hapten Linkers in Vitro To Select Hapten-Conjugate Vaccine Candidates for Pharmacokinetic Evaluation in Vivo.

PubMed

Arutla, Viswanath; Leal, Joseph; Liu, Xiaowei; Sokalingam, Sriram; Raleigh, Michael; Adaralegbe, Adejimi; Liu, Li; Pentel, Paul R; Hecht, Sidney M; Chang, Yung

2017-05-08

Since the demonstration of nicotine vaccines as a possible therapeutic intervention for the effects of tobacco smoke, extensive effort has been made to enhance nicotine specific immunity. Linker modifications of nicotine haptens have been a focal point for improving the immunogenicity of nicotine, in which the evaluation of these modifications usually relies on in vivo animal models, such as mice, rats or nonhuman primates. Here, we present two in vitro screening strategies to estimate and predict the immunogenic potential of our newly designed nicotine haptens. One utilizes a competition enzyme-linked immunoabsorbent assay (ELISA) to profile the interactions of nicotine haptens or hapten-protein conjugates with nicotine specific antibodies, both polyclonal and monoclonal. Another relies on computational modeling of the interactions between haptens and amino acid residues near the conjugation site of the carrier protein to infer linker-carrier protein conjugation effect on antinicotine antibody response. Using these two in vitro methods, we ranked the haptens with different linkers for their potential as viable vaccine candidates. The ELISA-based hapten ranking was in an agreement with the results obtained by in vivo nicotine pharmacokinetic analysis. A correlation was found between the average binding affinity (IC 50 ) of the haptens to an anti-Nic monoclonal antibody and the average brain nicotine concentration in the immunized mice. The computational modeling of hapten and carrier protein interactions helps exclude conjugates with strong linker-carrier conjugation effects and low in vivo efficacy. The simplicity of these in vitro screening strategies should facilitate the selection and development of more effective nicotine conjugate vaccines. In addition, these data highlight a previously under-appreciated contribution of linkers and hapten-protein conjugations to conjugate vaccine immunogenicity by virtue of their inclusion in the epitope that binds and activates B cells.

An experiment to study the effects of space flight cells of mesenchymal origin in the new model 3D-graft in vitro

NASA Astrophysics Data System (ADS)

Volova, Larissa

One of the major health problems of the astronauts are disorders of the musculoskeletal system, which determines the relevance of studies of the effect of space flight factors on osteoblastic and hondroblastic cells in vitro. An experiment to study the viability and proliferative activity of cells of mesenchymal origin on culture: chondroblasts and dermal fibroblasts was performed on SC "BION -M" No. 1 with scientific equipment " BIOKONT -B ." To study the effect of space flight conditions in vitro at the cellular level has developed a new model with 3D- graft as allogeneic demineralized spongiosa obtained on technology Lioplast ®. For space and simultaneous experiments in the laboratory of the Institute of Experimental Medicine and Biotechnology Samara State Medical University were obtained from the cell culture of hyaline cartilage and human skin, which have previously been grown, and then identified by morphological and immunohistochemical methods. In the experiment, they were seeded on the porous 3D- graft (controlled by means of scanning electron and confocal microscopy) and cultured in full growth medium. After completion of the flight of spacecraft "BION -M" No. 1 conducted studies of biological objects using a scanning electron microscope (JEOL JSM-6390A Analysis Station, Japan), confocal microscopy and LDH - test. According to the results of the experiment revealed that after a 30- day flight of the cells not only retained vitality, but also during the flight actively proliferate, and their number has increased by almost 8 times. In synchronous experiment, all the cells died by this date. The experimentally confirmed the adequacy of the proposed model 3D- graft in studying the effect of space flight on the morphological and functional characteristics of cells in vitro.