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BACKGROUND: With the increasing use of nanomaterials, the need for methods and assays to examine their immunosafety is becoming urgent, in particular for nanomaterials that are deliberately administered to human subjects (as in the case of nanomedicines). To obtain reliable results, standardised in vitro immunotoxicological tests should be used to determine the effects of engineered nanoparticles on human immune responses. However, before assays can be standardised, it is important that suitable methods are established and validated. RESULTS: In a collaborative work between European laboratories, existing immunological and toxicological in vitro assays were tested and compared for their suitability to test effects of nanoparticles on immune responses. The prototypical nanoparticles used were metal (oxide) particles, either custom-generated by wet synthesis or commercially available as powders. Several problems and challenges were encountered during assay validation, ranging from particle agglomeration in biological media and optical interference with assay systems, to chemical immunotoxicity of solvents and contamination with endotoxin. CONCLUSION: The problems that were encountered in the immunological assay systems used in this study, such as chemical or endotoxin contamination and optical interference caused by the dense material, significantly affected the data obtained. These problems have to be solved to enable the development of reliable assays for the assessment of nano-immunosafety.
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Bioensaio/métodos , Bioensaio/normas , Células/imunologia , Fatores Imunológicos/imunologia , Nanopartículas Metálicas , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Células/citologia , Células Cultivadas , Humanos , Interleucina-8/genética , Interleucina-8/imunologia , Nanopartículas Metálicas/efeitos adversos , Nanopartículas Metálicas/química , Regiões Promotoras Genéticas , Reprodutibilidade dos Testes , SolventesRESUMO
Nanoparticulate contrast agents for molecular imaging have attracted widespread interest for diagnostic applications with high resolution in medicine. Here we introduce polymer-based multifunctional nanoparticles exhibiting a near-infrared absorption in the range of the Nd:YAG laser wavelength of 1064 nm as a novel resorbable photoacoustic (PA) contrast system and report about their biological evaluation. Submicron-sized spherical nanoparticles with a high encapsulation efficiency (>87%) were created by incorporation of near-infrared dyes (IR5/IR26) in poly[(rac-lactide)-co-glycolide] (PLGA) with 50 mol% glycolide content via a specific spray-drying process in good yield (>75%). Subsequent application of a centrifugation protocol produced two different size fractions with diameters in the ranges 445-540 nm and 253-305 nm; these were further used for investigation of PA properties and cytotoxic effects. The prepared PLGA nanoparticles exhibited PA properties using a Nd:YAG laser-based system. After exposure of particle concentrations up to 10 µg·ml(-1) for 2 days no effects on viability, mitochondrial activity and proliferation, and cell death of human hepatocarcinoma cells and monkey kidney cells were observed. The excellent PA properties in combination with the positive biological results qualify the dye-loaded PLGA particles as promising candidates for a resorbable PA contrast system. FROM THE CLINICAL EDITOR: Photoacoustics (PA), a new modality, in which laser light is shined into tissue and absorbed by inherent proteins or synthetic particles is reflected back and received as ultrasound. This technique was shown to be effective with an erodible polymer particle containing near infrared dyes. In vitro, the PA properties of the PLGA particles persisted for 2 days in cell culture.
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Meios de Contraste/administração & dosagem , Portadores de Fármacos/química , Ácido Láctico/química , Nanopartículas/química , Ácido Poliglicólico/química , Acústica , Animais , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Células Hep G2 , Humanos , Ácido Láctico/efeitos adversos , Lasers de Estado Sólido , Microscopia Eletrônica de Varredura , Nanopartículas/efeitos adversos , Nanopartículas/ultraestrutura , Tamanho da Partícula , Ácido Poliglicólico/efeitos adversos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Espectroscopia de Luz Próxima ao Infravermelho , Ultrassonografia , Células VeroRESUMO
Non-invasive single cell analyses are increasingly required for the medicaldiagnostics of test substances or the development of drugs and therapies on the single celllevel. For the non-invasive characterisation of cells, impedance spectroscopy whichprovides the frequency dependent electrical properties has been used. Recently,microfludic systems have been investigated to manipulate the single cells and tocharacterise the electrical properties of embedded cells. In this article, the impedance ofpartially embedded single cells dependent on the cellular behaviour was investigated byusing the microcapillary. An analytical equation was derived to relate the impedance ofembedded cells with respect to the morphological and physiological change ofextracellular interface. The capillary system with impedance measurement showed afeasibility to monitor the impedance change of embedded single cells caused bymorphological and physiological change of cell during the addition of DMSO. By fittingthe derived equation to the measured impedance of cell embedded at different negativepressure levels, it was able to extrapolate the equivalent gap and gap conductivity betweenthe cell and capillary wall representing the cellular behaviour.
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Electric fields can be used for the characterisation and manipulation of single biological cells. One approach to avoid the effect of electrode polarisation is to position cells on micro holes and to apply the electrical fields via the micro holes. For a correct characterisation and optimal manipulation, the electrical properties of the micro hole/cell interface must be understood. In this article, the electrical characteristics of a micro hole-based cell chip were investigated. By FEM simulation, it was estimated that the impedance measurement with micro hole-based chip is most dependent on the cell adhesion/spread rather than the intra-cellular space (contribution of intra-cellular space to the total impedance: 0.07% at 1 kHz, 0.3% at 1 MHz). The effective frequency range in which the impedance related with cell state on the hole considerably influences total measured impedance was below several kiloHertz. From the experiments, it was shown that the impedance of cell cultured on the hole at the low frequency range is increased during the increase of cultivation period, but is sensitively decreased after applying only several nanolitres of culture medium including 5% dimethlysulfoxide. This micro hole-based chip has a potential for monitoring the cell growth and the membrane integrity of even single cell without any labelling.
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Técnicas Biossensoriais/métodos , Proliferação de Células/efeitos dos fármacos , Análise Espectral/métodos , Células Cultivadas , Dimetil Sulfóxido/farmacologia , Impedância ElétricaRESUMO
Use of balloon impedance catheters (BIC) for the characterization of plaques in vessels can support an optimal medical treatment of plaques. The sensitivity of impedance diagnoses with BIC is related with the distribution of electric fields determined by the electrode configuration. Using the three-dimensional finite element method (FEM) simulation, it was estimated how the relative positions of electrode array to the lipid in the vessel affect on the total impedance magnitude. Further, the short-circuiting effect was investigated with respect to the separation distance on the angular axis between the electrode arrays of angular set. By aid of FEM simulations, it is possible to design the sets of multielectrode arrays which have an optimized resolution for individual vessels.
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Arteriosclerose/diagnóstico , Arteriosclerose/fisiopatologia , Cateterismo Cardíaco/métodos , Diagnóstico por Computador/métodos , Impedância Elétrica , Eletrodos Implantados , Modelos Cardiovasculares , Animais , Simulação por Computador , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tomografia/métodosRESUMO
Use of impedance catheters can provide additional information about the composition and the morphology of early plaques in arteries. However, for a correct interpretation of the impedance data recorded inside a vessel, the extra-vessel conditions should not influence the measurement results. In this paper, we estimate the influence of the extra-vessel conditions on the impedance measurement of a vessel wall by using FEM simulation and a two-layer model. Therefore sensitivity fields are simulated. The simulations are validated by experiments and compared to analytical solutions. Further, the influence of the inner radius of a vessel on the measurement result is determined by FEM simulations. From experiments based on the two-layer model, it is found that the apparent resistance depends on the thickness of the first layer and the separation distance of the electrode structure. The measured result corresponds to the results of the FEM simulations, whereas the analytical solution assuming point electrodes is different from the measurement and simulation results. Under the assumption of homogenous and linear volume conductors, the FEM simulated distributions of sensitivity fields are determined. The inner diameter of the artery has no influence on the measurement results. The FEM simulation can support the design of electrode configuration and geometries for impedance catheters.
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Artérias/fisiopatologia , Estenose das Carótidas/diagnóstico , Estenose das Carótidas/fisiopatologia , Diagnóstico por Computador/métodos , Impedância Elétrica , Microeletrodos , Modelos Cardiovasculares , Pletismografia de Impedância/instrumentação , Animais , Simulação por Computador , Desenho Assistido por Computador , Desenho de Equipamento , Humanos , Pletismografia de Impedância/métodosRESUMO
Biotechnology demands powerful methods for the functional characterisation and monitoring of molecular alterations in tissues in response to various stimuli. Currently, cellular biosensors provide information about cell and tissue internal transduction pathways. In this article, recent biosensor systems are briefly described and the use of 3D tissue aggregates as recognition elements is discussed. An example of an innovative approach for drug testing using 3D heart muscle aggregates, as well as tumor models, positioned in capillary systems for electrical potential recording and impedance measurement is described. The effectiveness of drugs and therapies can be tested and monitored in a short time using such biohybrid sensors.
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Técnicas Biossensoriais/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Modelos Biológicos , Potenciais de Ação , Animais , Técnicas Biossensoriais/tendências , Capilares/citologia , Capilares/fisiologia , Avaliação Pré-Clínica de Medicamentos/tendências , Impedância Elétrica , Humanos , Miocárdio/citologia , Miocárdio/metabolismo , Resultado do TratamentoRESUMO
There is a clear need for cell- and tissue-based test systems for in vitro diagnostics and therapy evaluation. The advantages of tissue-based test systems are that the complex cell response is taken into account and that proteins are in their natural environment. The capillary measurement cell enables impedance measurement of small tissue samples with negligible electrode impedances and a high constant shunt resistance so that biologically relevant tissue parameters are determinable.
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Fenômenos Fisiológicos Celulares , Tecido Conjuntivo/fisiologia , Técnicas de Cultura/instrumentação , Impedância Elétrica , Eletroquímica/instrumentação , Ação Capilar , Técnicas de Cultura/métodos , Eletroquímica/métodos , Desenho de EquipamentoRESUMO
BACKGROUND: Newer techniques are required to identify atherosclerotic lesions that are prone to rupture. Electric impedance spectroscopy (EIS) is able to provide information about the cellular composition of biological tissue. The present study was performed to determine the influence of inflammatory processes in type Va (lipid core, thick fibrous cap) and Vc (abundant fibrous connective tissue while lipid is minimal or even absent) human atherosclerotic lesions on the electrical impedance of these lesions measured by EIS. METHODS AND RESULTS: EIS was performed on 1 aortic and 3 femoral human arteries at 25 spots with visually heavy plaque burden. Severely calcified lesions were excluded from analysis. A highly flexible micro-electrode mounted onto a balloon catheter was placed on marked regions to measure impedance values at 100 kHz. After paraffin embedding, visible marked cross sections (n = 21) were processed. Assessment of lesion types was performed by Movats staining. Immunostaining for CD31 (marker of neovascularisation), CD36 (scavenger cells) and MMP-3 (matrix metalloproteinase-3) was performed. The amount of positive cells was assessed semi-quantitatively. 15 type Va lesions and 6 type Vc lesions were identified. Lesions containing abundant CD36-, CD31- and MMP-3-positive staining revealed significantly higher impedance values compared to lesions with marginal or without positive staining (CD36 + 455 ± 50 Ω vs. CD36- 346 ± 53 Ω, p = 0.001; CD31 + 436 ± 43 Ω vs. CD31- 340 ± 55 Ω, p = 0.001; MMP-3 + 400 ± 68 Ω vs. MMP-3- 323 ± 33 Ω, p = 0.03). CONCLUSIONS: Atherosclerotic lesions with abundant neovascularisation (CD31), many scavenger receptor class B expressing cells (CD36) or high amount of MMP-3 immunoreactivity reveal significantly higher impedance values compared to lesions with marginal or no detection of immunoreactivity. Findings suggest that inflammatory processes in vulnerable plaques affect the impedance of atherosclerotic lesions and might therefore be detected by EIS.
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Angiografia/métodos , Aterosclerose/diagnóstico , Aterosclerose/patologia , Espectroscopia Dielétrica/métodos , Procedimentos Endovasculares/métodos , Aorta/patologia , Antígenos CD36/análise , Tecido Conjuntivo/patologia , Artéria Femoral/patologia , Humanos , Masculino , Metaloproteinase 3 da Matriz/análise , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análiseRESUMO
For studying cellular processes three-dimensional (3D) in vitro models are of a high importance. For tissue engineering approaches osseous differentiation is performed on 3D scaffolds, but material depending influences promote cellular processes like adhesion, proliferation and differentiation. To investigate developmental processes of mesenchymal stem cells without cell-substrate interactions, self-contained in vitro models mimicking physiological condition are required. However, with respect to scientific investigations and pharmaceutical tests, it is essential that these tissue models are well characterised and are of a high reproducibility. In order to establish an appropriate in vitro model for bone formation, different protocols are compared and optimised regarding their aggregate formation efficiency, homogeneity of the aggregates, the viability and their ability to induce differentiation into the osteogenic lineage. The protocols for the generation of 3D cell models are based on rotation culture, hanging drop technique, and the cultivation in non adhesive culture vessels (single vessels as well as 96 well plates). To conclude, the cultivation of hMSCs in 96 well non adhesive plates facilitates an easy way to cultivate homogenous cellular aggregates with high performance efficiency in parallel. The size can be controlled by the initial cell density per well and within this spheroids, bone formation has been induced.
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Células-Tronco Mesenquimais/citologia , Modelos Biológicos , Osteogênese , Engenharia Tecidual/métodos , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Highly-sensitive analysis systems based on cellular multi-parameter are needed in the diagnostics. Therefore we improved our previously developed chip platform for another additional analysis method, the fluorescence in situ hybridization. Fluorescence in situ hybridization (FISH) is a technique used in the diagnostics to determine the localization and the presence or absence of specific DNA sequence. To improve this labor- and cost-intensive method, we reduced the assay consumption by a factor of 5 compared to the standard protocol. Microhole chips were used for making the cells well addressable. The chips were fabricated by semiconductor technology on the basis of a Silicon wafer with a thin deposited silicon nitride layer (Si(3)N(4)). Human retina pigment epithelia (ARPE-19) cells were arrayed on 5-µm holes of a 35 × 35 microhole-array by a gently negative differential pressure of around 5 mbar. After 3 hours of incubation the cells were attached to the chip and the FISH protocol was applied to the positioned cells. A LabView software was developed to simplify the analysis. The software automatically counts the number of dots (positive labeled chromosome regions) as well as the distance between adjacent dots. Our developed platform reduces the assay consumption and the labor time. Furthermore, during the 3 hours of incubation non-invasive or minimal-invasive methods like Raman- and impedance-spectroscopy can be applied.
Assuntos
Hibridização in Situ Fluorescente/métodos , Técnicas Analíticas Microfluídicas/métodos , Linhagem Celular , Fluoresceína-5-Isotiocianato/química , Humanos , Processamento de Imagem Assistida por Computador , Luz , Microscopia de Fluorescência , Software , Xantenos/químicaRESUMO
There is a lack of methods suitable for generation of data about the dynamics of effects on cell membranes with a high sensitivity. Such methods are urgently needed to support the optimisation of interaction of substances, particles or materials with cell. The goal of this article is to use an improved microhole chip system to monitor the alterations of cells due to the interactions of polymer-DNA complexes. This should demonstrate exemplarily that subtoxic effect of biological relevant particles or substances at relevant concentrations can be monitored for several hours. By using a microhole cell chip and a microfluidic unit single cells can be electrically interfaced via microholes and the use of small electrodes with high impedances is not necessary. For separation and positioning of the cells onto the hole negative pressure is applied on the reverse side of the chip. Under cell culture conditions the cell starts to spread on the biocompatible insulating chip membrane resulting in a stable interface to an adherent growing cell. After the spreading process is finished, the polymer/polyplex solution is added and the impedance is measured with respect to time. To illustrate the cellular parameter which can affect the measured impedance a simple simulation based on the finite element method (FEM) is performed. It was shown for the first time that the impedance-based method predicated on the microhole chip can be used for biological relevant substances at relevant concentrations and that it is more sensitive than the well-established biological marker.
Assuntos
Técnicas Biossensoriais/métodos , Membrana Celular/efeitos dos fármacos , Técnicas Analíticas Microfluídicas/métodos , Células Cultivadas , Impedância Elétrica , HumanosRESUMO
INTRODUCTION: Nanoparticulate contrast agents are of great interest for diagnostic applications with high resolution in medicine. Here we present polymer-based degradable nanoparticles exhibiting a near infrared (NIR) absorption suitable for photoacoustic imaging. METHODS: The nanoparticles were prepared by incorporation of indocyanine green (ICG) as NIR dye in poly[(rac-lactide)-co-glycolide] (PLGA) via an optimized spray drying process. By application of a multi-step centrifugation protocol, two different size fractions were achieved. The biocompatibitilty of the nanoparticles was tested in 2D cell cultures (human hepatocarcinoma cells and monkey kidney cells) using WST-1, BrdU and LDH assay. RESULTS: Spherical particles were obtained with a good yield (>81%), showing a high NIR-dye encapsulation efficiency (>98%). By multi-step centrifugation, two different size fractions with a mean diameter of 640 nm and 390 nm were obtained. Cytotoxicity studies of the synthesized ICG-loaded PLGA particles were performed. No cytotoxic effect on metabolic activity, proliferation, or membrane integrity was observed. CONCLUSION: The high optical absorption at the relevant NIR-wavelength around 800 nm in combination with absence of cytotoxicity qualifies the ICG-loaded PLGA particles as promising candidates for degradable photoacoustic contrast agents.
Assuntos
Corantes/química , Meios de Contraste/química , Verde de Indocianina/química , Nanopartículas , Poliglactina 910/química , Espectroscopia de Luz Próxima ao Infravermelho , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Corantes/toxicidade , Meios de Contraste/toxicidade , Metabolismo Energético/efeitos dos fármacos , Células Hep G2 , Humanos , Verde de Indocianina/toxicidade , Tamanho da Partícula , Poliglactina 910/toxicidade , Propriedades de Superfície , Células VeroRESUMO
In most conventional in vitro toxicological assays, the response of a complete cell population is averaged, and therefore, single-cell responses are not detectable. Such averaging might result in misinterpretations when only individual cells within a population respond to a certain stimulus. Therefore, there is a need for non-invasive in vitro systems to verify the toxicity of nanoscale materials. In the present study, a micro-sized cell culture chamber with a silicon nitride membrane (0.16 mm2) was produced for cell cultivation and the detection of specific cell responses. The biocompatibility of the microcavity chip (MCC) was verified by studying adipogenic and neuronal differentiation. Thereafter, the suitability of the MCC to study the effects of nanoparticles on a small cell population was determined by using a green fluorescence protein-based reporter cell line. Interleukin-8 promoter (pIL8) induction, a marker of an inflammatory response, was used to monitor immune activation. The validation of the MCC-based method was performed using well-characterized gold and silver nanoparticles. The sensitivity of the new method was verified comparing the quantified pIL8 activation via MCC-based and standard techniques. The results proved the biocompatibility and the sensitivity of the microculture chamber, as well as a high optical quality due to the properties of Si3N4. The MCC-based method is suited for threshold- and time-dependent analysis of nanoparticle-induced IL8 promoter activity. This novel system can give dynamic information at the level of adherent single cells of a small cell population and presents a new non-invasive in vitro test method to assess the toxicity of nanomaterials and other compounds.PACS: 85.35.Be, 81.16.Nd, 87.18.Mp.
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Subretinal implants aim to replace the photoreceptor function in patients suffering from degenerative retinal disease by topically applying electrical stimuli in the subretinal space. Critical obstacles in the design of high-resolution subretinal implants include the proximity of stimulating electrodes to the target cells and enabling nutrient flow between the retina and the choroid. The present work evaluates the adhesion, migration and survival of retinal cells on an ultrathin (5 µm), highly porous (Ø 1 µm spaced 3 µm), gelatin-coated polyimide (PI) membrane. The biocompatibility was examined in mice indicating a good tolerance upon subcutaneous implantation with only a mild inflammatory response. In addition, organotypic cultures of rat retina evidenced that the porous membrane allowed the necessary nutrient flow for the retinal cell survival and maintenance. A transscleral implantation technique was applied to position the membrane into the subretinal space of rats. The effect on the obtained retinal integration was investigated in vivo using scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT). In 12 out of 18 rat eyes, the implant was successfully placed subretinally. SLO and OCT demonstrated complete retinal attachment and fluorescein angiography showed no retinal vascular abnormalities over and around the implant, immediately after and up to four weeks after the implantation. Histological examination of the eyes showed a close attachment of a thin fibrocyte layer to the implant, the occlusion of the pores by living cells and the survival of some photoreceptors at the implantation site.
Assuntos
Membranas Artificiais , Implantação de Prótese/métodos , Retina/cirurgia , Animais , Adesão Celular , Sobrevivência Celular , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Técnicas de Cultura de Órgãos , Ratos , Ratos Wistar , Retina/citologia , Retina/ultraestrutura , Tomografia de Coerência ÓpticaRESUMO
Lab-on-a-chip systems are increasingly applied in cell-based assays for toxicology and drug testing. In this paper, an on-chip integrated lensless microscopy module using a direct projection method for optical monitoring of the shadow images of adherent growing mammalian cells is presented. The biological cells are conserved and interfaced by a microfabricated cavity chip with a 1 microm thick silicon nitride (Si(3)N(4)) substrate onto the surface of a 5 megapixel CMOS image sensor with 2.2 microm pixel size. The optical resolution of the assembly is estimated by the contact/proximate printing theory from optical lithography. Further characterization is made by imaging microbeads in chips with the Si(3)N(4)-membrane as well as in cavity chips with membranes made from dry film resist (DFR, thickness 20, 40 and 60 microm). The module represents a 3 × optical microscope for cell morphology imaging. The function is demonstrated by the growth monitoring of L929 cells cultured in cavity chips with Si(3)N(4) substrate for 2 days and by checking the colorimetric staining of cells with a compromised membrane.
Assuntos
Fibroblastos/citologia , Fibroblastos/fisiologia , Aumento da Imagem/instrumentação , Iluminação/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Microscopia/instrumentação , Processamento de Sinais Assistido por Computador/instrumentação , Animais , Adesão Celular , Linhagem Celular , Proliferação de Células , Desenho de Equipamento , Análise de Falha de Equipamento , Lentes , Camundongos , Reprodutibilidade dos Testes , Semicondutores , Sensibilidade e Especificidade , Integração de SistemasRESUMO
Human mesenchymal stem cells are promising candidates for cell-based therapies since they have the capacity to differentiate into a variety of cell types. However, the acceptance of hMSCs for clinical applications as well as in vitro tissue models will depend on strategies for standard characterisations. Impedance spectroscopy is a proven and powerful tool for non-invasive monitoring of cellular processes. The aim of this study was to prove the hypothesis, that the process of osteogenic differentiation can be monitored non-invasively and time-continuously by using impedance spectroscopy. This hypothesis was examined for 2D cell layers of hMSCs by continuous impedance spectroscopy employing a planar electrode-based chip and for 3D aggregates of hMSCs after 21 and 25 days of osteogenic treatment by using a capillary measurement system. The impedance spectra of osteogenic treated hMSCs reported a significant increase of the magnitude of impedance compared to controls cultivated in normal growth medium. The osteogenic status of the cells was determined by alkaline phosphatase expression and von Kossa staining. In respect to that finding it is concluded that impedance spectroscopy is an appropriate method for non-invasive characterisation of osteogenic differentiation of hMSCs, which is relevant for quality control of cell-based implants and cell-based test systems for drug development.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Osteogênese/fisiologia , Análise Espectral/métodos , Idoso , Agregação Celular , Células Cultivadas , Impedância Elétrica , Humanos , Células-Tronco Mesenquimais/metabolismoRESUMO
Gadolinium hydroxide, Gd(OH)(3), nanostructures were examined for their possible use in imaging and tracking of cells and tissues by investigating their cellular interactions and cytotoxic behaviors. For this purpose, Gd(OH)(3) nanorods (length, several hundred nanometers; diameter, approximately 40 nm) and spherical nanoparticles (average diameter, <10 nm) were synthesized by solvothermal decomposition of gadolinium containing molecular precursors. After comprehensive characterization of material properties, human colon adenocarcinoma (Caco2) and human lung epithelial (A549) cells were incubated with Gd(OH)(3) nanostructures in concentrations up to 900 microg/mL to perform cytotoxicity assays (BrdU (5-bromo-2'-deoxyuridine), WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzol-disulfonate)) and living/dead staining. As observed in all in vitro assays, the investigated Gd(OH)(3) nanostructures do not induce any significant cytotoxic effect, making them promising candidates for a new class of contrast agents, which may overcome the limitations of organic stains such as photobleaching and single usage.
Assuntos
Gadolínio/toxicidade , Nanoestruturas , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Gadolínio/química , HumanosRESUMO
In this study the critical parameters directing osteogenic differentiation of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) were investigated, key factors and conditions identified and improved protocols for a more cell-type adapted differentiation developed. Today only little information about the specific conditions directing osteogenic development is available and current protocols for cultivation and differentiation of UCB-MSCs are based mainly on experience with bone marrow-derived MSCs (BM-MSCs) without further adaptation. Thus, protocols for improved osteoinduction are of particular interest. The goal of this study was to investigate the influence of three different culture media (A) alpha MEM, 15% FBS, (B) DMEM, 15% FBS and (C) MSCGM, 10% SingleQuot growth supplement on the osteogenic differentiation of UCB-MSCs. Moreover, a systematic analysis of two concentrations of dexamethasone (10(-8)M/10(-7)M) in combination with or without BMP-2 (10(-7)M) was carried out by detecting the expression of alkaline phosphatase (ALP), collagen-1 and the mineralization of ECM. We found that MSCGM, 10% SingleQuot had a supportive effect on the osteogenic differentiation of UCB-MSCs. In case of treatment with 10(-8)M dexamethasone, mineralization occurred in combination with BMP-2 exclusively, while a concentration of 10(-7)M dexamethasone led to a high amount of mineralized ECM and the expression of collagen-1 independent of BMP-2 addition. According to this data dexamethasone is the leading osteoinductive factor, but BMP-2 seems to have supportive properties in UCB-MSCs. In conclusion, MSCGM supplemented with 10% SingleQuot and 10(-7)M dexamethasone was the condition identified to be best for inducing the osteogenic differentiation of UCB-MSCs.
Assuntos
Diferenciação Celular/fisiologia , Meios de Cultura , Sangue Fetal/citologia , Células-Tronco Mesenquimais/citologia , Osteogênese/fisiologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Técnicas de Cultura de Células/métodos , Divisão Celular/fisiologia , Humanos , Recém-Nascido , Células-Tronco Mesenquimais/enzimologiaRESUMO
Pesticides used to control unwanted insects are potentially toxic to humans. In assessing the risk involved in exposure to pesticides or complex chemical mixtures, an in vitro cell-based test can provide useful information regarding danger to human health. Cell differentiation is a biological process of fundamental importance in developing and adult organisms. In this paper, we propose a cell-based test system for continuous, label-free monitoring of the effect of test substances on stem cell differentiation. Using a prefabricated electrode-based chip and impedance measurement system, we investigated the influence of chlorpyrifos (a pesticide) on the differentiation of human mesenchymal stem cells (hMSCs) to adipocytes. The state of hMSCs on electrodes during adipogenic differentiation or after application of the cytotoxic substance was clearly reflected in the impedance measurement. Chlorpyrifos caused a partially uncovered electrode area with a decreased number of lipid vacuoles, thus leading to a rapid decrease in resistance in the cell layer. After removal of the chlorpyrifos, the cell layer resistance was regained due to the renewed covering of the electrodes by hMSCs. However, an increase in lipid vacuoles was not observed. From this, it was concluded that the measured resistance of hMSCs is determined by the electrical properties in the extra cellular space (e.g., cell/electrode or cell/cell gap), but not by the lipid vacuoles appearing in intracellular space during adipogenic differentiation.