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)

Evaluatie van MOF-geïnduceerde veranderingen in celgedrag door elektrische celsubstraatdetectie

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.

Auteur in de schijnwerpers: vooruitgang bij het evalueren van de reactie van menselijke longepitheelcellen op metaal-organische raamwerken 

Elektrische cel-substraatdetectie voor real-time evaluatie van toxicologische profielen van metaal-organische kaders

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)  

Elektrische celsubstraatimpedantie detectie (ECIS)

Neem om te beginnen een 96-putplaat met interdigitated vingerverbindingselektroden die een gebied van ongeveer vier vierkante millimeter beslaan. Plaats de plaat op het putstation en klik op de installatieknop. Als de putten goed zijn aangesloten, verschijnen er groene metingen.Alle niet-aangesloten putten worden geïdentificeerd als rood. Voeg 200 microliter media toe aan elke put die wordt gebruikt en plaats de plaat vervolgens in de elektrische celsubstraatimpedantiedetectie of ECIS-station. Om de putten te stabiliseren en voor te bereiden op experimenten, klikt u op instellen, gevolgd door controleren en klikt u vervolgens op stabiliseren.Verwijder na 40 minuten de plaat uit het station en verwijder de media uit elke put. Zaai de B2B-cellen met een dichtheid van 40.000 cellen per milliliter in de putten met de cellen en plaats de plaat op het ECIS-station in de incubator. Klik vervolgens op controleren om ervoor te zorgen dat alle elektroden worden gelezen door de sensoren op het station.Selecteer meerdere frequenties om elke elektrode op zeven verschillende frequenties te meten. Klik op start en laat het 24 uur draaien, zodat de cellen een confluente monolaag kunnen vormen. De meting van elke put wordt in een andere kleur weergegeven.Na ongeveer 24 uur zullen de putjes met cellen een verhoogde weerstand op de monitor vertonen, wat de bevestiging van de cellen aan de elektroden aangeeft. Druk op pauze om het verzamelen van gegevens te onderbreken. Bijvoorbeeld voor mediawijzigingen, reagentia-editie, enzovoort.Stel de ZIF8-nanodeeltjes bloot aan de lege put en de celputten met een volume van 100 microliter per put, plaats de plaat op het station en klik nogmaals op controleren om ervoor te zorgen dat de sensoren alle elektroden lezen. Klik op hervatten om de experimenten na de gekozen pauze te hervatten. Klik na het verzamelen van de gegevens op Voltooien om de gegevens op te slaan als een spreadsheetbestand.Om de alfaparameters te krijgen, klikt u op model en klikt u op het pop-upscherm op media alleen goed en selecteert u vervolgens de put met alleen media. Klik op referentie instellen voor gegevensevaluatie en selecteer de referentie met betrekking tot het besturingsexperiment. Klik op zoeken om de referentiefitting te starten.Selecteer instellen en vervolgens T-bereik aanpassen om de gegevens ten opzichte van de geselecteerde referentie te verwerken. Als u de celbijlage wilt evalueren, selecteert u alfa. Nadat het systeem de analyse heeft voltooid, klikt u ten slotte op RBA opslaan en slaat u de parameters op als een spreadsheetbestand.De analyse van het celgedrag bij blootstelling aan ZIF8 MOFs werd uitgevoerd door middel van elektrische celsubstraatimpedantiedetectie. De beelden toonden veranderingen in de interacties tussen het celsubstraat en de veranderingen zijn afhankelijk van het tijdstip van blootstelling en de dosis die wordt gebruikt.

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 ...