RESUMO
Marine biotoxins in fish and shellfish can cause several symptoms in consumers, such as diarrhea, amnesia, or even death by paralysis. Monitoring programs are in place for testing shellfish on a regular basis. In some countries testing is performed using the so-called mouse bioassay, an assay that faces ethical concerns not only because of animal distress, but also because it lacks specificity and results in high amounts of false positives. In Europe, for lipophilic marine biotoxins (LMBs), a chemical analytical method using LC-MS/MS was developed as an alternative and is now the reference method. However, safety is often questioned when relying solely on such a method, and as a result, the mouse bioassay might still be used. In this study the use of a cell-based assay for screening, i.e., the neuro-2a assay, in combination with the official LC-MS/MS method was investigated as a new alternative strategy for the detection and quantification of LMBs. To this end, samples that had been tested previously with the mouse bioassay were analyzed in the neuro-2a bioassay and the LC-MS/MS method. The neuro-2a bioassay was able to detect all LMBs at the regulatory levels and all samples that tested positive in the mouse bioassay were also suspect in the neuro-2a bioassay. In most cases, these samples contained toxin levels (yessotoxins) that explain the outcome of the bioassay but did not exceed the established maximum permitted levels.
Assuntos
Alternativas aos Testes com Animais/métodos , Bioensaio/métodos , Toxinas Marinhas/análise , Intoxicação por Frutos do Mar/prevenção & controle , Frutos do Mar/análise , Alternativas aos Testes com Animais/instrumentação , Animais , Bioensaio/instrumentação , Bivalves , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Reações Falso-Positivas , Toxinas Marinhas/toxicidade , Camundongos , Venenos de Moluscos , Oxocinas/análise , Oxocinas/toxicidade , Frutos do Mar/toxicidade , Intoxicação por Frutos do Mar/etiologia , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodosRESUMO
Performing drug screening of tissue derived from cancer patient biopsies using physiologically relevant 3D tumour models presents challenges due to the limited amount of available cell material. Here, we present a microfluidic platform that enables drug screening of cancer cell-enriched multicellular spheroids derived from tumour biopsies, allowing extensive anticancer compound screening prior to treatment. This technology was validated using cell lines and then used to screen primary human prostate cancer cells, grown in 3D as a heterogeneous culture from biopsy-derived tissue. The technology enabled the formation of repeatable drug concentration gradients across an array of spheroids without external fluid actuation, delivering simultaneously a range of drug concentrations to multiple sized spheroids, as well as replicates for each concentration. As proof-of-concept screening, spheroids were generated from two patient biopsies and a panel of standard-of-care compounds for prostate cancer were tested. Brightfield and fluorescence images were analysed to provide readouts of spheroid growth and health, as well as drug efficacy over time. Overall, this technology could prove a useful tool for personalised medicine and future drug development, with the potential to provide cost- and time-reduction in the healthcare delivery.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Triagem em Larga Escala/métodos , Neoplasias/tratamento farmacológico , Alternativas aos Testes com Animais/instrumentação , Alternativas aos Testes com Animais/métodos , Antineoplásicos/uso terapêutico , Biópsia , Desenvolvimento de Medicamentos/métodos , Ensaios de Seleção de Medicamentos Antitumorais/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Dispositivos Lab-On-A-Chip , Microfluídica/instrumentação , Microfluídica/métodos , Neoplasias/patologia , Cultura Primária de Células/instrumentação , Cultura Primária de Células/métodos , Estudo de Prova de Conceito , Reprodutibilidade dos Testes , Esferoides Celulares/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Although initially developed to replace animal testing in drug development, human 'organ on a chip' (organ chip) microfluidic culture technology offers a new tool for studying tissue development and pathophysiology, which has brought us one step closer to carrying out human experimentation in vitro In this Spotlight article, I discuss the central role that developmental biology played in the early stages of organ-chip technology, and how these models have led to new insights into human physiology and disease mechanisms. Advantages and disadvantages of the organ-chip approach relative to organoids and other human cell cultures are also discussed.
Assuntos
Biologia do Desenvolvimento , Dispositivos Lab-On-A-Chip , Microfluídica , Organoides/citologia , Engenharia Tecidual , Alternativas aos Testes com Animais/instrumentação , Alternativas aos Testes com Animais/métodos , Biologia do Desenvolvimento/instrumentação , Biologia do Desenvolvimento/métodos , Biologia do Desenvolvimento/tendências , Doença , Desenvolvimento Embrionário/fisiologia , Humanos , Microfluídica/instrumentação , Microfluídica/métodos , Microfluídica/tendências , Modelos Biológicos , Técnicas de Cultura de Órgãos/métodos , Técnicas de Cultura de Órgãos/tendências , Esferoides Celulares/citologia , Engenharia Tecidual/métodos , Engenharia Tecidual/tendênciasRESUMO
ABSTRACT The use of a commercial kit for the monocyte-activation test (MAT) was evaluated for assessing pyrogenic contamination of hyperimmune sera . Three batches of sera, two pyrogen free and one pyrogenic, were tested. Endotoxin spike recover indicated that sample dilutions from 1/2 to 1/10 are suitable. Kit transport and storage conditions were also evaluated, proving that an adequate cold chain must be assured to achieve good results. Furthermore, the commercial MAT kit seemed suitable to replace the rabbit pyrogen test (RPT) for pyrogen testing of hyperimmune sera, although further tests are needed to a full validation.
Assuntos
Pirogênios/análise , Soro , Kit de Reagentes para Diagnóstico , Monócitos/classificação , Alternativas aos Testes com Animais/instrumentaçãoRESUMO
Alternative methods and their use in planning and conducting toxicology experiments have become essential for modern toxicologists, thus reducing or replacing living animals. Although in vitro human co-culture models allow the establishment of biologically relevant cell-cell interactions that recapitulate the tissue microenvironment and better mimic its physiology, the number of publications is limited specifically addressing this scientific area and utilizing this test method which could provide an additional valuable model in toxicological studies. In the present study, an in vitro model based on central nervous system (CNS) cell co-cultures was implemented using a transwell system combining human neuronal cells (SH-SY5Y cell line) and glial cells, namely astrocytes (D384 cell line), to investigate neuroprotection of D384 on SH-SY5Y and vice versa. The model was applied to test acute (24-48 hours) cytotoxicity of 3 different neurotoxicants: (1) methyl mercury (1-2.5 µM), (2) Fe3O4 nanoparticles (1-100 µg/mL), and (3) methylglyoxal (0.5-1 mM). Data were compared to mono-cultures evaluating the mitochondrial function and cell morphology. The results clearly showed that all compounds tested affected the mitochondrial activity and cell morphology in both mono-culture and co-culture conditions. However, astrocytes, when cultured together with neurons, diminish the neurotoxicant-induced cytotoxic effects that occurred in neurons cultured alone, and astrocytes become more resistant in the presence of neurons. This human CNS co-culture system seems a suitable cell model to feed high-throughput acute screening platforms and to evaluate both human neuronal and astrocytic toxicity and neuroprotective effects of new and emerging materials (eg, nanomaterials) and new products with improved sensitivity due to the functional neuron-astrocyte metabolic interactions.
Assuntos
Alternativas aos Testes com Animais/métodos , Astrócitos/efeitos dos fármacos , Técnicas de Cocultura/métodos , Neurônios/efeitos dos fármacos , Xenobióticos/toxicidade , Alternativas aos Testes com Animais/instrumentação , Astrócitos/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura/instrumentação , Humanos , Nanopartículas de Magnetita/toxicidade , Compostos de Metilmercúrio/toxicidade , Microscopia de Contraste de Fase , Mitocôndrias/efeitos dos fármacos , Neurônios/patologia , Aldeído Pirúvico/toxicidade , Testes de Toxicidade AgudaRESUMO
Body-on-a-chip systems replicate the size relationships of organs, blood distribution and blood flow, in accordance with human physiology. When operated with tissues derived from human cell sources, these systems are capable of simulating human metabolism, including the conversion of a prodrug to its effective metabolite, as well as its subsequent therapeutic actions and toxic side-effects. The system also permits the measurement of human tissue electrical and mechanical reactions, which provide a measure of functional response. Since these devices can be operated with human tissue samples or with in vitro tissues derived from induced pluripotent stem cells (iPS), they can play a significant role in determining the success of new pharmaceuticals, without resorting to the use of animals. By providing a platform for testing in the context of human metabolism, as opposed to animal models, the systems have the potential to eliminate the use of animals in preclinical trials. This article will review progress made and work achieved as a direct result of the 2015 Lush Science Prize in support of animal-free testing.
Assuntos
Alternativas aos Testes com Animais/instrumentação , Dispositivos Lab-On-A-Chip , Células CACO-2 , Sobrevivência Celular , Técnicas de Cocultura , Células HT29 , Humanos , Farmacocinética , Testes de ToxicidadeRESUMO
In toxicology, the strategies for testing the hazardous potential of substances are changing as a result of the ongoing progress in the development of in vitro methods and the demand of the authorities to reduce animal testing. Even in the complex field of inhalation toxicology with its high requirements on the technical implementation and cell culture models, the preconditions for using such methods are fulfilled. We here introduce a sophisticated technique that enables the stable and reproducible exposure of cultivated cells to airborne substances at the air-liquid interface by means of the CULTEX(®) Radial Flow System (RFS) module. The feasibility and suitability of the experimental setup is demonstrated by dose-response investigations of mainstream cigarette smoke and particulate matter of four substances in different lung epithelial cell lines. A dose-dependent cytotoxcity of the test substances was verified by applying different exposure times. The high reproducibility of the results indicate the reliability of the presented method and recommend the integration of such in vitro approaches in the field of inhalation toxicology by advancing their regulatory validation.
Assuntos
Poluentes Atmosféricos/toxicidade , Alternativas aos Testes com Animais/métodos , Técnicas de Cultura de Células/métodos , Exposição por Inalação , Testes de Toxicidade/métodos , Poluentes Atmosféricos/química , Alternativas aos Testes com Animais/instrumentação , Técnicas de Cultura de Células/instrumentação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Desenho de Equipamento , Estudos de Viabilidade , Humanos , Modelos Biológicos , Material Particulado/química , Material Particulado/toxicidade , Transição de Fase , Reprodutibilidade dos Testes , Poluição por Fumaça de Tabaco/efeitos adversos , Testes de Toxicidade/instrumentaçãoRESUMO
Measurement of ocular irritancy is a necessary step in the safety evaluation of both industrial and consumer products. Assessment of the acute eye irritation potential is therefore part of the international regulatory requirements for testing of chemicals. The Bovine Corneal Opacity and Permeability (BCOP) assay is generally accepted as a valid in vitro alternative method to the Draize eye irritation test to detect corrosive and severe eye irritants (category 1), but has not proven sensitive enough to discriminate accurately moderate (category 2A/2B) to mild and non-irritating compounds. In the currently accepted BCOP assay, opacity is determined by the amount of light transmission through the cornea, and permeability is determined by the amount of sodium fluorescein dye that passes through all corneal cell layers. Both measurements are used to assign an In Vitro Irritancy Score (IVIS) for prediction of the in vivo ocular irritation potential of a test substance. Nowadays, opacity is measured by an OP-KIT opacitometer providing a center-weighted reading of light transmission by measuring changes in voltage when the transmission of white light passes through the cornea alters. As a consequence, this may underestimate opacity that develops as spots or heterogeneous opaque areas on the periphery of an isolated cornea. A prototype of a laser light-based opacitometer (PLLBO) allowing better measurement of opacities was developed by Van Goethem et al. (2010). This new device showed improved sensitivity to detect subtle changes in corneal transparency. Furthermore, the new opacitometer allowed the analysis of the complete corneal surface and was able to detect more efficiently opaque spots located along the sides of the excised corneas. A further improved prototype of the PLLBO was constructed in combination with a camera and a speckle noise reducer. Treatment conditions of the corneas in the cornea holders were optimized in order to mimic more the real in vivo situation. A set of test compounds with irritancy potencies especially in the mild and moderate range was tested. The improved LLBO showed some promising features which potentially could improve the usefulness of the BCOP test. Adaptation of cornea holders showed to be of limited value and only restricted to concentrations up to 15% which mimics more test conditions in industry. This 3-year research project was sponsored by the Stavros Niarchos Foundation (Greece).
Assuntos
Alternativas aos Testes com Animais/métodos , Bioensaio/métodos , Córnea/efeitos dos fármacos , Irritantes/toxicidade , Alternativas aos Testes com Animais/instrumentação , Animais , Bioensaio/instrumentação , Bovinos , Córnea/metabolismo , Opacidade da Córnea/induzido quimicamente , Desenho de Equipamento , Técnicas In Vitro , Lasers , Permeabilidade/efeitos dos fármacos , CoelhosRESUMO
BACKGROUND: In biomedical research, the past two decades have seen the advent of in vitro model systems based on stem cells, humanized cell lines, and engineered organotypic tissues, as well as numerous cellular assays based on primarily established tumor-derived cell lines and their genetically modified derivatives. OBJECTIVE: There are high hopes that these systems might replace the need for animal testing in regulatory toxicology. However, despite increasing pressure in recent years to reduce animal testing, regulators are still reluctant to adopt in vitro approaches on a large scale. It thus seems appropriate to consider how we could realistically perform regulatory toxicity testing using in vitro assays only. DISCUSSION AND CONCLUSION: Here, we suggest an in vitro-only approach for regulatory testing that will benefit consumers, industry, and regulators alike.
Assuntos
Alternativas aos Testes com Animais/métodos , Poluentes Ambientais/toxicidade , Regulamentação Governamental , Testes de Toxicidade/métodos , Alternativas aos Testes com Animais/instrumentação , Alternativas aos Testes com Animais/legislação & jurisprudência , Alternativas aos Testes com Animais/normas , Animais , Ecotoxicologia/instrumentação , Ecotoxicologia/métodos , Ecotoxicologia/normas , Humanos , Testes de Toxicidade/instrumentação , Testes de Toxicidade/normasRESUMO
BACKGROUND AND AIM OF THE STUDY: During the development of a mechanical heart valve prosthesis, many studies are conducted to guarantee its correct function. Currently, investigations into the thrombogenic potential of a valve after its replacement are conducted with expensive and time-consuming chronic animal trials. Hence, the study aim was to develop and test an alternative system to resolve such thrombogenic issues. METHODS: The Thrombosis Tester of the Helmholtz Institute Aachen (THIA II) has a reasonably small priming volume (220-270 ml) that allows analysis of the thrombogenic potential of two valves, using one human blood bottle. RESULTS: Hydrodynamic evaluation demonstrated an absolutely stable physiological pressure and flow progression at the aortic and pulmonary positions. A sinus geometry of the human aortic root is implemented downstream of the valve in order to guarantee physiological leaflet motion. The tester remained absolutely thrombus-free during several tests carried out with minimally anticoagulated porcine blood, while the valves showed reproducible thrombus formation in reasonable locations. Tests with fully heparinized porcine blood showed that a soft silicon fixture for the valve could reduce hemolysis in the THIA II. CONCLUSION: This in-vitro test protocol can enable the optimization of a valve design during the early stages of its research and development. The system can provide a unique and suitable supplement to animal trials for testing thrombogenic performance, under constant and reproducible boundary conditions, including considerable physiological and pathological circumstances such as the influence of valve position (aortic, pulmonic), and a comparison of different valve types.
Assuntos
Alternativas aos Testes com Animais/instrumentação , Próteses Valvulares Cardíacas/efeitos adversos , Trombose/etiologia , Coagulação Sanguínea , Desenho de Equipamento , Humanos , Hidrodinâmica , Teste de Materiais , Trombose/prevenção & controleRESUMO
Data on eye irritation are generally needed for the hazard identification of chemicals. As the Bovine Corneal Opacity and Permeability (BCOP) test has been accepted by many regulatory agencies for the identification of corrosive and severe ocular irritants since September 2009 (OECD Test Guideline 437, TG 437), we evaluated this alternative method for routine testing at BASF. We demonstrated our technical proficiency by testing the reference standards recommended in TG 437, and 21 additional materials with published BCOP and in vivo data. Our results matched the published in vitro data very well, but with some intentionally selected false negatives (FNs) and false positives (FPs), the concordance was 77% (24/31), with FN and FP rates of 20% (2/10) and 24% (5/21), respectively. In addition, we tested 21 in-house materials, demonstrating the utility of the BCOP assay for our own test material panel. Histopathological assessment of the corneas by light microscopy was also conducted, as this was suggested as a means of improving the identification of FNs. The histopathology corrected the classification of some FNs, but also increased the number of FPs. Parallel to the test method evaluation, we compared three new opacitometer models with the current standard device. We recommend the use of an opacitometer developed in our BASF laboratory, which has certified components and electronic data storage, resulting in what we consider to be excellent sensitivity, stability and reproducibility.
Assuntos
Alternativas aos Testes com Animais , Cáusticos/toxicidade , Opacidade da Córnea/induzido quimicamente , Epitélio Corneano/efeitos dos fármacos , Irritantes/toxicidade , Alternativas aos Testes com Animais/instrumentação , Animais , Bovinos , Opacidade da Córnea/metabolismo , Opacidade da Córnea/patologia , Equipamentos para Diagnóstico , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Permeabilidade , Reprodutibilidade dos TestesAssuntos
Alternativas aos Testes com Animais/métodos , Bioengenharia/métodos , Biomimética/métodos , Dispositivos Lab-On-A-Chip , Modelos Biológicos , Especificidade de Órgãos , Alternativas aos Testes com Animais/instrumentação , Animais , Bioengenharia/instrumentação , Biomimética/instrumentação , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Avaliação Pré-Clínica de Medicamentos/instrumentação , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/fisiologia , Fígado/fisiopatologia , Pulmão/irrigação sanguínea , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/fisiologia , Camundongos , Modelos Animais , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Reprodutibilidade dos Testes , Silício/químicaRESUMO
The development of aerosol medicines typically involves numerous tests on animals, due to the lack of adequate in vitro models. A new in vitro method for testing pharmaceutical aerosol formulations on cell cultures was developed, consisting of an aerosolisation unit fitting a commercial dry powder inhaler (HandiHaler(c), Boehringer Ingelheim, Germany), an air-flow control unit (Akita(c), Activaero, Germany) and a custom-made sedimentation chamber. This chamber holds three Snapwell(c) inserts with monolayers of pulmonary epithelial cells. The whole set-up, referred to as the Pharmaceutical Aerosol Deposition Device On Cell Cultures (PADDOCC) system, aims to mimic the complete process of aerosol drug delivery, encompassing aerosol generation, aerosol deposition onto pulmonary epithelial cells and subsequent drug transport across this biological barrier, to facilitate the investigation of new aerosol formulations in the early stages of development. We describe here, the development of the design and the protocol for this device. By testing aerosol formulations of budesonide and salbutamol sulphate, respectively, reproducible deposition of aerosol particles on, and the integrity of, the pulmonary cell monolayer could be demonstrated.
Assuntos
Alternativas aos Testes com Animais/métodos , Técnicas de Cultura de Células/métodos , Aerossóis , Alternativas aos Testes com Animais/instrumentação , Animais , Linhagem Celular , Células Cultivadas/citologia , Células Cultivadas/fisiologia , Cromatografia Líquida de Alta Pressão , Desenho de Equipamento , Humanos , Reprodutibilidade dos TestesRESUMO
In vitro culture models that employ human liver cells could be potent tools for predictive studies on drug toxicity and metabolism in the pharmaceutical industry. A bioreactor culture model was developed that permits the three-dimensional co-culture of liver cells under continuous medium perfusion with decentralised mass exchange and integral oxygenation. We tested the ability of the system to support the long-term maintenance and differentiation of primary human liver cells. The effects of the initial cell quality were investigated by comparing cultures from resected, non-preserved liver with cultures from liver graft tissue damaged by long-term preservation. In cultures originating from non-preserved liver, protein and urea synthesis, glucose metabolism, and cytochrome (CYP450) activities were stable over the 2-week culture period, with maximal activities at the end of the first week in culture. Enzyme induction led to increased 7-ethoxyresorufin O-deethylase activities of up to 20 times the basal value. In cultures from preservation-damaged liver, recovery of metabolic activities was detected during bioreactor culture. After two weeks, most biochemical parameters approached those of cultures from non-preserved human liver. Light microscopy demonstrated the three-dimensional reorganisation of hepatocytes and non-parenchymal cells in co-culture. Long-term maintenance, and even the regeneration of specific functional activities of human liver cells, can be achieved in the bioreactor. This could facilitate the introduction into the pharmaceutical industry of in vitro drug testing with primary human liver cells.
Assuntos
Alternativas aos Testes com Animais/métodos , Reatores Biológicos , Hepatócitos/citologia , Fígado/citologia , Albuminas/metabolismo , Alternativas aos Testes com Animais/instrumentação , Aspartato Aminotransferases/metabolismo , Dióxido de Carbono/metabolismo , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Técnicas de Cocultura , Citocromo P-450 CYP1A1/metabolismo , Glucose/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Lidocaína/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Transplante de Fígado , Preservação de Órgãos , Oxigênio/metabolismo , Testosterona/metabolismo , Ureia/metabolismoRESUMO
The bovine cornea opacity and permeability assay (BCOP) has been in use for nearly 10 years but has not been submitted for regulatory approval. In previous reports we have presented corneal hydration and endothelial damage as additional endpoints in this assay and have suggested that the design of the BCOP's corneal holder should be modified. The standard holder used in the BCOP assay induces physical damage to the cornea because it contacts clear cornea causing edge damage to the epithelial, stromal and endothelial layers. Second, by forcing a curved, oval-shaped bovine cornea into a flat, circular opening, corneal wrinkling occurs which can alter the cornea's optical characteristics and, most importantly, induces endothelial damage. We now report on a redesigned BCOP corneal holder that clamps onto the sclera, maintains normal corneal shape and does not cause damage to the endothelium. This ensures that irritancy tests are conducted using healthy, anatomically normal tissue. Tests of this holder using acetone, trichloroacetic acid, isopropanol and benzalkonium chloride show that it is now possible to evaluate effects of chemical substances on the endothelium. The effects of these compounds on corneal opacity and hydration in the new holder are similar to their effects on the cornea in the standard holder.
Assuntos
Alternativas aos Testes com Animais/instrumentação , Córnea/citologia , Opacidade da Córnea , Equipamentos e Provisões , Animais , Bovinos , Permeabilidade da Membrana Celular , Córnea/patologia , Opacidade da Córnea/etiologia , Opacidade da Córnea/patologia , Epitélio Corneano/citologia , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/patologia , Irritantes/toxicidade , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
We present a new in vitro assay for screening of potential teratogens, based on staining of cultured mouse fibroblastoid L929 cells for the determination of number of live and dead cells and of cell morphology, employing automatic video recording, followed by detection of the stained specimen and calculation of endpoint values by the use of a computerized microscope workstation. Ten different parameters were combined empirically into a single index describing general alterations in cell morphology, and, subsequently, measurements of alterations in morphology and proliferation were combined to produce a single empirical index aimed at predicting teratogenic potency. The assay was employed in two different laboratories on 10 coded compounds; 7 compounds that have demonstrated in vivo teratogenic potentials: valproic acid (VPA), pentyl-4-yn-VPA, retinoic acid (RA), 13-cis-RA, AM580, thalidomide, and alpha-EM12 and 3 compounds for which no teratogenic potential has been demonstrated: isobutyl-4-yn-VPA, phytanic acid, and beta-EM12. Within each of the three groups of compounds the nonteratogens generally caused smaller alterations in cell morphology than the teratogens, although the effects of thalidomide and related compounds generally were minor or insignificant. The data support the hypothesis that cell morphology and proliferation in combination with other endpoints may be employed for in vitro screenings of potential teratogens, although studies of additional compounds are needed in order to establish the general validity of the procedure.
Assuntos
Alternativas aos Testes com Animais/métodos , Teratogênicos/toxicidade , Alternativas aos Testes com Animais/instrumentação , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia de Vídeo/instrumentação , Microscopia de Vídeo/métodos , Teratogênicos/classificaçãoRESUMO
Cell-vessel wall interactions (adhesion, emigration) and cell-cell cohesion (aggregation) have been assessed primarily in animal experiments. The cell function analyser (CFA) is an in vitro vascular model, in which the three components of Virchow's triad are present in a highly standardised and variable form. The CFA permits visual and quantitative analysis of cellular adhesion, emigration and aggregation under physiologically relevant flow conditions (i.e. to the arteries and to the microcirculation). Although the method does not entail the use of a living animal or of animal tissue, as is true for animal experiments, with the CFA specimen fixation and histomorphological analysis after the experiment is possible. The efficacy of the method for platelet function testing has been verified by numerous clinical studies. The wide variability of test parameters make CFA suitable for in vitro analysis of other cell-vessel-wall-mediated processes, such as inflammation, wound healing and tumour metastasis. We present: 1) a description of the CFA method and underlying hemodynamic principles, 2) a review of clinical and experimental results with platelets and, 3) the first results of convective flow-mediated leukocyte-endothelial interactions. The CFA provides an in vitro alternative to animal experiments, can be classified as a replacement method and possesses an analysis spectrum that will greatly reduce the overall need for the previous.