Name: Electrical Cell-Substrate Impedance Sensing (ECIS)
Uploaded: 2023-05-26T12:30:00
Description: Watch this Scientific Journal Video about Electrical Cell-Substrate Impedance Sensing ECIS at JoVE.com

electrical cell-substrate impedance sensing (ecis)

Evaluación de los cambios en el comportamiento de las células inducidos por MOF mediante la detección eléctrica del sustrato de la célula

Metal-organic frameworks (MOFs) are hybrids formed through the combination of metal ions and organic linkers1,2 in organic solvents. Due to the variety of such combinations, MOFs possess structural diversity3, tunable porosity, high thermal stability, and high surface areas4,5. Such characteristics make them attractive candidates in a variety of applications, from gas storage6,7 to catalysis8,9, and from contrast agents10,11 to drug delivery units12,13. However, the implementation of MOFs into such applications has raised concerns relative to their safety to both the users and the environment. Preliminary studies have shown, for instance, that cellular function and growth change upon the exposure of cells to metal ions or linkers used for MOF synthesis1,14,15. For instance, Tamames-Tabar et al. demonstrated that ZIF-8 MOF, a Zn-based MOF, was leading to more cellular changes in a human cervical cancer cell line (HeLa) and a mouse macrophage cell line (J774) relative to Zr-based and Fe-based MOFs. Such effects were&#160;presumably due to the metal component of ZIF-8 (i.e., Zn), which could potentially induce cell apoptosis upon framework disintegration and Zn ion release1. Similarly, Gandara-Loe et al. demonstrated that HKUST-1, a Cu-based MOF, caused the highest reduction in mouse retinoblastoma cell viability when used at concentrations of 10 &#181;g/mL or greater. This was presumably due to the Cu metal ion incorporated during the synthesis of this framework, which, once released, could induce oxidative stress in the exposed cells15.
Moreover, analysis showed that the exposure&#160;to MOFs with different physicochemical characteristics could lead to varying responses of exposed cells. For instance, Wagner et al. demonstrated that ZIF-8 and MIL-160 (an Al-based framework), used in the exposure of an immortalized human bronchial epithelial cell, led to cellular responses dependent on frameworks&#39; physicochemical properties, namely hydrophobicity, size, and structural characteristics16. Complementarily, Chen et al. demonstrated that a concentration of 160 &#181;g/mL MIL-100(Fe) exposed to human normal liver cells (HL-7702) caused the largest loss in cellular viability, presumably due to the metal component of this specific framework (i.e., Fe17).
While these studies categorize MOFs&#39; deleterious effects on cellular systems based on their physicochemical characteristics and exposure concentrations, thus raising potential concerns with framework implementation, especially in biomedical fields, most of these evaluations are based on single time point colorimetric assays. For instance, it was shown that when (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) and water-soluble tetrazolium salt (WST-1) assays were used, these biochemical reagents could lead to false positives upon&#160;their&#160;interactions with the particles that the cells were also exposed to18. The tetrazolium salt and neutral red reagents were&#160;shown to possess a high adsorption or binding affinity onto the surfaces of the particles, resulting to agent signal interference19. Moreover, for other types of assays, such as flow cytometry, which was previously shown to be used for assessing changes in cells exposed to MOFs20,21, it was shown that major issues have to be circumvented if a viable analysis of particles&#39; deleterious effects is to be considered. In particular, detection ranges of the particles&#39; sizes, especially in mixed populations like the ones offered by MOFs or references of the particles used for calibration before cellular changes, have to be addressed22. It was also shown that the dye used during cell labeling for such cytometry assays could also interfere with the nanoparticles that the cells were exposed to23.
The goal of this study was to use a real-time, high-throughput evaluation assay to assess changes in cell behavior upon exposure to a select MOF. Real-time evaluations can help provide insights into time-dependent effects, as related to the windows of exposures16. Further, they provide information on changes in cell-substrate interactions, cell morphology, and cell-cell interactions, as well as how such changes depend on the physicochemical properties of the materials of interest and exposure times24,25 respectively.
To demonstrate the validity and applicability of the proposed approach, human bronchial epithelial (BEAS-2B) cells, ZIF-8 (a hydrophobic framework of zeolitic imidazolate16), and electric cell-substrate impedance sensing (ECIS) were used. BEAS-2B cells represent a model for lung exposure26 and have been previously used to evaluate changes upon the exposure of cells to nanoclays and their thermally degraded byproducts26,27,28,&#160;as well as assess the toxicity of nanomaterials, such as single-walled carbon nanotubes (SWCNTs)18. Furthermore, such cells have been used for more than 30 years as a model for pulmonary epithelial function29. ZIF-8 was chosen due to its wide implementation&#160;in catalysis30 and as contrast agents31 for bioimaging and drug delivery32, and thus for the extended potential for lung exposure during such applications. Lastly, ECIS, the noninvasive, real-time technique, was previously used to evaluate changes in cell adherence, proliferation, motility, and morphology16,26 as a result of a variety of interactions between analytes (both materials and drugs) and exposed cells in real-time16,18,28. ECIS uses an alternating current (AC) to measure the impedance of cells immobilized on gold electrodes, with the impedance changes giving insights into changes in resistance and capacitance at the&#160;cell-gold substrate interface, barrier function as induced by cell-cell interactions, and over-cell layer coverage of such gold electrodes33,34. Using ECIS allows quantitative measurements at a nanoscale resolution in a noninvasive, real-time manner26,34.
This study assesses and compares the simplicity and ease of evaluation of MOF-induced changes in cellular behavior in real-time with single-point assay evaluations. Such a study could be further extrapolated for evaluating cell profiles in response to exposure to other particles of interest, thus allowing for safe-by-design particle testing and subsequent helping with&#160;implementation. Moreover, this study could complement genetic and cellular assays that are single-point evaluations. This could lead to a more informed analysis of the deleterious effects of particles on the cellular population and could be used for screening such particles&#39; toxicity in a high-throughput manner16,35,36.

Autor destacado: Avances en la evaluación de la respuesta de las células epiteliales pulmonares humanas a las estructuras metal-orgánicas 

Detección eléctrica de sustratos de células para la evaluación en tiempo real de perfiles toxicológicos de estructuras metal-orgánicas

My research aims to evaluate changes in human lung epithelial cells'behavior upon exposure to metal organic framework, hybrids formed by combining metal ions in organic linkers in organic solvents. This research demonstrated that the physical and chemical composition of metal organic frameworks, as well as their cellular exposure ratios, affects the behavior of human lung epithelial cells in a time and dose-dependent manner. This can be recorded in a high throughput in real time fashion.Our research helps to evaluate, understand the mechanisms of toxicity of metal organic frameworks upon exposure to cellular systems. The current method allows continuous cell monitoring in a real time and in high throughput manner. Additionally, this method does not rely on any external reagents, and thus it reduces any possible interference they could have with the frameworks used for exposure.Our laboratory will continue to focus on understanding and evaluating the mechanisms of toxicity associated with nanomaterials exposures, as well as using such information to synthesize new and safe systems that could be implemented in biomedical applications at minimum risks to the user or the environment.

Watch this Scientific Journal Video about Electrical Cell-Substrate Impedance Sensing ECIS at JoVE.com

Electrical Cell-Substrate Impedance Sensing (ECIS)  

Detección de impedancia de sustrato de celda eléctrica (ECIS)

Para comenzar, tome una placa de 96 pocillos que contiene electrodos de conexión de dedos interdigitados que cubren un área de aproximadamente cuatro milímetros cuadrados. Coloque la placa en la estación de pozos y haga clic en el botón de configuración. Si los pozos están conectados correctamente, aparecerán lecturas verdes.Todos los pozos no conectados se identificarán en rojo. Agregue 200 microlitros de medio a cada pocillo que se utilice y luego coloque la placa en la estación de detección de impedancia del sustrato de la celda eléctrica o ECIS. Para estabilizar los pozos y prepararlos para los experimentos, haga clic en configuración, seguido de verificación y, a continuación, haga clic en estabilizar.Después de 40 minutos, retire la placa de la estación y retire el medio de cada pocillo. Siembre las células B2B a una densidad de 40.000 células por mililitro en los pocillos que contienen las células y coloque la placa en la estación ECIS de la incubadora. A continuación, haga clic en comprobar para asegurarse de que todos los electrodos sean leídos por los sensores de la estación.Seleccione el tiempo de frecuencia múltiple para medir cada electrodo en siete frecuencias diferentes. Haga clic en iniciar y deje que se ejecute durante 24 horas para que las células puedan formar una monocapa confluente. La lectura de cada pozo se muestra en un color diferente.Después de aproximadamente 24 horas, los pocillos que contienen celdas mostrarán una mayor resistencia en el monitor, lo que indica la unión de las celdas a los electrodos. Presione pausa para interrumpir la recopilación de datos. Por ejemplo, para cambios de medios, edición de componentes, etc.Exponga las nanopartículas ZIF8 al pocillo en blanco y a los pocillos celulares con un volumen de 100 microlitros por pocillo, coloque la placa en la estación y haga clic en verificar nuevamente para asegurarse de que los sensores estén leyendo todos los electrodos. Haga clic en reanudar para reanudar los experimentos después de la pausa elegida. Después de la recopilación de datos, haga clic en finalizar para guardar los datos como un archivo de hoja de cálculo.Para obtener los parámetros alfa, haga clic en el modelo y, en la pantalla emergente, haga clic en solo el pozo de medios, luego seleccione el pozo que solo tiene medios. Haga clic en establecer referencia para la evaluación de datos y seleccione la referencia relacionada con el experimento de control. Haga clic en buscar para iniciar el accesorio de referencia.Seleccione establecer y, a continuación, ajuste el rango T para procesar los datos relativos a la referencia seleccionada. Para evaluar la conexión de celda, seleccione alfa. Finalmente, una vez que el sistema finalice el análisis, haga clic en guardar RBA y guarde los parámetros como un archivo de hoja de cálculo.El análisis del comportamiento de la célula tras la exposición a los MOF ZIF8 se realizó mediante la detección de impedancia del sustrato eléctrico de la célula. Las imágenes mostraron cambios en las interacciones entre el sustrato celular y los cambios dependen del tiempo de exposición y de la dosis que se utilice.

Research

JoVE Journal

Bioengineering

Electrical Cell-Substrate Impedance Sensing (ECIS)

Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of ...