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)

Evaluating MOF-Induced Cell Behavior Changes by Electric Cell-Substrate Sensing

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.

Author Spotlight: Advances in Evaluating Human Lung Epithelial Cells' Response to Metal-Organic Frameworks 

Registrering av elektrisk cellesubstrat for sanntidsevaluering av toksikologiske profiler for metallorganisk rammeverk

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)  

Registrering av elektrisk celle-substratimpedans (ECIS)

For å begynne, ta en 96 brønnplate som inneholder interdigitated fingerforbindelseselektroder som dekker et område på omtrent fire kvadratmillimeter. Plasser platen på brønnstasjonen og klikk på oppsettsknappen. Hvis brønnene er riktig tilkoblet, vises grønne avlesninger.Eventuelle ikke-tilkoblede brønner vil bli identifisert som røde. Legg til 200 mikroliter media til hver brønn som brukes, og plasser deretter platen i den elektriske cellesubstratimpedansfølingen, eller ECIS-stasjonen. For å stabilisere brønnene og klargjøre dem for eksperimenter, klikk oppsett, etterfulgt av sjekk, og klikk deretter på stabiliser.Etter 40 minutter, fjern platen fra stasjonen og fjern mediet fra hver brønn. Frø B2B-cellene med en tetthet på 40.000 celler per milliliter inn i brønnene som inneholder cellene, og plasser platen på ECIS-stasjonen i inkubatoren. Klikk deretter på sjekk for å sikre at alle elektrodene leses av sensorene på stasjonen.Velg flere frekvenstider for å måle hver elektrode ved syv forskjellige frekvenser. Klikk på start og la den kjøre i 24 timer slik at cellene kan danne et sammenflytende monolag. Hver brønns lesing vises i en annen farge.Etter ca. 24 timer vil brønnene som inneholder celler vise økt motstand på monitoren som indikerer at cellene festes til elektrodene. Trykk på pause for å avbryte datainnsamlingen. For eksempel for medieendringer, reagensutgaver og så videre.Utsett ZIF8 nanopartiklene til den tomme brønnen og cellebrønnene med et volum på 100 mikroliter per brønn, Plasser platen på stasjonen og klikk på sjekk igjen for å sikre at sensorene leser alle elektrodene. Klikk på fortsett for å gjenoppta eksperimentene etter den valgte pausen. Etter datainnsamlingen klikker du fullfør for å lagre dataene som en regnearkfil.For å få alfaparametrene, klikk på modellen, og på den dukket opp skjermen, klikk bare på media godt, og velg deretter brønnen som bare har media. Klikk på angi referanse for dataevaluering og velg referansen relatert til kontrolleksperimentet. Klikk på finn for å starte referansetilpasningen.Velg sett og tilpass deretter T-området for å behandle dataene i forhold til den valgte referansen. Hvis du vil evaluere cellevedlegget, velger du alfa. Til slutt, etter at systemet er ferdig med analysen, klikker du lagre RBA og lagrer parametrene som en regnearkfil.Analysen av celleoppførselen ved eksponering for ZIF8 MOFs ble utført gjennom registrering av elektrisk cellesubstratimpedans. Bildene viste endringer i cellesubstratinteraksjonene, og endringene er avhengig av eksponeringstidspunktet og dosen som brukes.

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 ...