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)

Valutazione dei cambiamenti di comportamento delle celle indotte da MOF mediante rilevamento del substrato della cella elettrica

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.

Autore in primo piano: Progressi nella valutazione della risposta delle cellule epiteliali polmonari umane alle strutture metallo-organiche 

Rilevamento elettrico cellula-substrato per la valutazione in tempo reale dei profili tossicologici della struttura metallo-organica

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)  

Rilevamento elettrico dell'impedenza cella-substrato (ECIS)

Per iniziare, prendi una piastra a 96 pozzetti contenente elettrodi di connessione a dito interdigitati che coprono un'area di circa quattro millimetri quadrati. Posizionare la piastra sulla stazione del pozzo e fare clic sul pulsante di configurazione. Se i pozzetti sono collegati correttamente, appariranno le letture verdi.Tutti i pozzi non collegati saranno identificati come rossi. Aggiungere 200 microlitri di terreno a ciascun pozzetto utilizzato, quindi posizionare la piastra nel rilevamento dell'impedenza del substrato della cella elettrica o nella stazione ECIS. Per stabilizzare i pozzetti e prepararli per gli esperimenti, fare clic su Imposta, seguito da Verifica, quindi fare clic su Stabilizza.Dopo 40 minuti, rimuovere la piastra dalla stazione e rimuovere il materiale da ciascun pozzetto. Seminare le cellule B2B a una densità di 40.000 cellule per millilitro nei pozzetti contenenti le cellule e posizionare la piastra sulla stazione ECIS nell'incubatrice. Quindi, fare clic su controlla per assicurarsi che tutti gli elettrodi vengano letti dai sensori sulla stazione.Selezionare il tempo a frequenza multipla per misurare ciascun elettrodo a sette frequenze diverse. Fare clic su Start e lasciarlo funzionare per 24 ore in modo che le celle possano formare un monostrato confluente. La lettura di ogni pozzetto viene visualizzata in un colore diverso.Dopo circa 24 ore, i pozzetti contenenti le celle mostreranno una maggiore resistenza sul monitor indicando l'attacco delle celle agli elettrodi. Premere pausa per interrompere la raccolta dei dati. Ad esempio, per le modifiche ai supporti, l'edizione dei reagenti e così via.Esporre le nanoparticelle di ZIF8 al pozzetto bianco e ai pozzetti delle celle con un volume di 100 microlitri per pozzetto, posizionare la piastra sulla stazione e fare nuovamente clic su controlla per assicurarsi che i sensori leggano tutti gli elettrodi. Fare clic su Riprendi per riprendere gli esperimenti dopo la pausa scelta. Dopo la raccolta dei dati, fare clic su Fine per salvare i dati come file di foglio di calcolo.Per ottenere i parametri alfa fare clic sul modello e, nella schermata visualizzata, fare clic su solo supporto, quindi selezionare il pozzo che contiene solo media. Fare clic su Imposta riferimento per la valutazione dei dati e selezionare il riferimento relativo all'esperimento di controllo. Fare clic su Trova per avviare il raccordo di riferimento.Selezionare Imposta e adatta intervallo T per elaborare i dati relativi al riferimento selezionato. Per valutare l'allegato della cella, selezionare alfa. Infine, al termine dell'analisi da parte del sistema, fate clic su Salva RBA (Save RBA) e salvate i parametri come file di foglio di calcolo.L'analisi del comportamento cellulare in seguito all'esposizione ai MOF ZIF8 è stata eseguita attraverso il rilevamento dell'impedenza del substrato elettrico della cella. Le immagini hanno mostrato cambiamenti nelle interazioni tra substrato cellulare e i cambiamenti dipendono dal tempo di esposizione e dalla dose utilizzata.

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 ...