• Save
Ecis Applied Biophysics
Upcoming SlideShare
Loading in...5
×
 

Ecis Applied Biophysics

on

  • 3,932 views

The ECIS is a turnkey system that provides researchers with an advanced, automated, non-invasive means to monitor cell behavior in real-time and without the use of labels.

The ECIS is a turnkey system that provides researchers with an advanced, automated, non-invasive means to monitor cell behavior in real-time and without the use of labels.

Statistics

Views

Total Views
3,932
Views on SlideShare
3,931
Embed Views
1

Actions

Likes
1
Downloads
51
Comments
0

1 Embed 1

http://www.slideshare.net 1

Accessibility

Categories

Upload Details

Uploaded via as Microsoft PowerPoint

Usage Rights

© All Rights Reserved

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Processing…
Post Comment
Edit your comment
  • Caderina calcium dependent

Ecis Applied Biophysics Ecis Applied Biophysics Presentation Transcript

  • ECIS  Electric Cell-substrate Impedance Sensing
  • ECIS measures the change in impedance of a small electrode to AC current flow . The heart of the measurement is a specialized slide that has 8 or 96 individual wells for cell culturing. The base of the device has an array of gold film electrodes that connect to the ECIS electronics to each of the 8 wells.
  • WE CE WE: Working Electrode CE: Counter Electrode The ECIS Electrodes 250 µm
  • An ECIS 96 well multiple electrode array
  • Without cells , the current flows unrestrained from the surface of the electrodes.
  • With cells attached and spread upon this region, the current must now flow in the spaces under and between the cells, as the cell membrane are essentially insulators.
  • ECIS  Electric Cell-substrate Impedance Sensing A cell morphology biosensor AC Current source ECIS electrode Counter electrode Culture medium (electrolyte) impedance measurement <4 mA/cm 2 4000 Hz The measurement is non-invasive
  • Start of the measurement - no cells attached - resistance is about 2000 ohms. Inoculation , cells anchor and spread on the base of the well including the active 250 µm electrode. With the presence of the cells , their insulating plasma membranes constrain the electrical current and force it to flow in regions beneath and between the cells. This convoluted current path causes large changes in the measured impedance. With the confluent cell layer in place , the resistance now has reached nearly 15,000 ohms. It is important to note that the AC current used in making these measurements (approximately 1 uA) and the resulting voltage drops across the cells (a few mV) has no detectable effects upon them; the measurement is non-invasive.
  • Cell Inoculation (10 5 cells per cm 2 ) ~50 cells on the active electrode NRK cells No cells Data is from a single 250  m dia. Electrode
  • low frequency low frequency AC Frequency determines Current Pathway high frequency
  • AC Frequency in ECIS = Focus Plane in Microscopy Microvilli Stress Fibers Actin Belt MDCK Cells stained with Phalloidin in CLSM
  • Cell-Matrix Contacts 40 kHz Cell-Cell Contacts 400 Hz MDCK Cells In Situ Analysis of Cell Junctions De Novo Formation of Cell-Matrix and Cell-Cell Contacts
  • Analysis of ECIS Raw Data Monitoring the Formation of a Cell Monolayer Cell Spreading 40 kHz MDCK From Joachim Wegener University of Regensburg
  • Analysis of ECIS Raw Data Monitoring the Formation of a Cell Monolayer Cell Spreading 40 kHz MDCK From Joachim Wegener University of Regensburg Barrier Formation 400 Hz
  • Monitoring Endothelial Barrier Function A published model fits the experimental data The measured impedance can be broken down into three parameters 1) Rb, the barrier function of the cell layer 2) Alpha , a term associated with the constricted current flow beneath the cell 3) Cm , the membrane capacitance [Giaever, I. and Keese, C.R., PNAS 81, 3761 (1991)]
  • CHO cells engineered to over-express the muscarinic receptor exposed to the agonist carbachol
  • Data analysis using the ECIS model  morphological information Treatment of CHO-M1T cells with carbachol
  •  
  • The 16 Well Array Station provides electrical contact for two 8 well ECIS arrays.
  • The 96 Well Array Station provides electrical contact for a single 96 well ECIS Array.
  • user-friendly, software interface allows versatility for data acquisition and analysis.
  • 16 Well Proliferation Impedance Data
  • 96 Well Proliferation Impedance Data
  •  
  • 96 Well Array Map Data
  •  
  •  
  • Wound Healing Assay ECIS is a fully automated wound healing assay that generates quantitative data in real time-with out opening the incubator door. ECIS measurements are label-free and highly reproducible . Replaces the traditional &quot; scratch &quot; or &quot; scrape &quot; assay. Instead of disrupting the cell layer mechanically with a toothpick, needle or pipette tip and following the migration of cells to &quot;heal&quot; the wound with a microscope, we employ electric signal s to both wound and monitor the healing process. ECIS electrical wounding is only directed at the small population of cells in contact with the active 250um diameter ECIS electrode, producing a well defined 250 um wound. Which can be verified both with the ECIS measurement and with vital staining. Unlike the traditional scrape method, with the ECIS Wound your protein coating is unaffected by the current , it remains fully intact.
  • Wound Healing Assay
  • Automated real-time wound-healing assay to measure various rates of migration.
  •  
  • Cell Attachment and Spreading One of the most direct ECIS measurements is that of the attachment and spreading behaviors of cells. These measurements allow one to study and quantify the interaction of cultured cells with extracellular matrix (ECM) proteins and other macromolecules continuously and in real time.
  • Different cell line behavior The graph shows the attachment and spreading behaviors of two different cell types -namely BSC1 (African Green Monkey Kidney) and NRK (Normal Rat Kidney) cells. Cells were inoculated in ECIS wells at time zero in sufficient number to form a confluent cell layer (100,000 cells per cm). Each cell line studies in this manner will have its own characteristic shape and final values. In these data, the electrodes were not treated in any special manner before the inoculation but are coated with proteins adsorbed from the fetal bovine serum in the culture medium.
  • MDCK II cells inoculated on electrodes pre-coated with various proteins FN fibronectin LAM laminin VN vitronectin BSA bovine serum albumin BSA FN Inoculation Confluent Cell-free Capacitance at high freq. measures the open (cell-free) electrode area Adsorbed proteins alter cell spreading dynamics Monitoring the dynamics of cell attachment & spreading. Capacitance plots are also used to determine and measure cell confluence and proliferation.
  • Used for general morphological changes in cells. Data shows attachment and spreading of cells on various absorbed protein layers.
  • ECIS Measurements of Metastatic Potential We have used the ability of ECIS to detect changes in cell morphology to design whole-cell assays related to the behavior of the cancer cell including metastatic potential.
  • ECIS Measurements of Metastatic Potential The lost of the resistance is due to the lost of integrity of the endothelial cell layer in response to the activities of the cancer cells.
  • Data shows the extravasation of an endothelial layer upon exposure to metastatic cell lines. Challenge with active and heat-killed cells The highly metastatic MLL cell line rapidly breaks down the resistance of the established endothelial cell layer, but the same cells when first heat killed (15 minutes at 56 degrees C) have no effect verifying that the assay is indeed seeing biological activities. In these data all additions are at time zero. challenge of HUVEC cell layers with several different human prostatic carcinoma cell line
  • Angiogenesis Overall, angiogenesis research starts with the monitoring of endothelial cells (barrier function, signaling, cell growth); these observations are then studied to understand their relationship to angiogenesis and tube formation Picture from &quot;Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement&quot; Garcia, 2001.
  • Angiogenesis ECIS is a highly useful in vitro assay to determine endothelial cell growth and its inhibition as well as the barrier function of endothelial monolayers . In preliminary ECIS experiments with VEGF and other growth factors and their inhibitors, results can commonly be correlated with angiogenesis inhibition in vivo. In some cases, the blocking of endothelial cell proliferation has actually been proven to encourage tube formation. Although ECIS cannot directly follow capillary formation in tumor masses, it can monitor the effect of tumor cells upon a normal endothelial cell monolayer in vitro. Monitoring barrier function (permeability), ECIS has successful recorded extravasion of endothelial cell layers by metastatic cells and hence can be used as a tool for testing anti-cancer therapies in vitro.
  • Barrier Function of Cell Layers Measurement of barrier function of cell layers in tissue culture via ECIS has proven to be a significant and rapidly growing use of this technology. Cell layers with very high TER (transcutaneus el. res.) values such as endothelial cells from the brain and epithelial cells exhibiting tight junctions can be measured electrically using porated membrane supports as well as with ECIS.
  • Note that for molecules to travel from the lower chamber to the space above the cell layer, they must also travel in the constricted space beneath the cells to reach the intercellular junctions. Because of this, one does not measure the true barrier function but rather a combination of the constricted paths beneath the cells and the transcellular pathway. The ECIS method is unique in being able to distinguish between these two pathways , since they each affect the measured resistance and capacitance monitored by ECIS in different ways. Using a  published mode l , the true barrier function due to intercellular junctions can be extracted from ECIS data. Barrier function Constricted path beneath cells
  • Mathematical modeling can be used to monitor changes in barrier function as shown above following the addition of VEGF to endothelial cells. Barrier Function Data
  • Cell Proliferation Measurements ECIS measurements can be used to monitor cell proliferation, and experiments can be designed to determine how various changes in cell and culture conditions affect the rates at which the cell monolayer approaches confluence. To accomplish these studies, arrays are inoculated with a low cell density providing room for the dividing cell population. As the cell number increases, the amount of electrode area covered with the spread cells grows accordingly, causing the electrode impedance to rise.
  • In Vitro Toxicology The ECIS approach furnishes data that are by nature quantitative , and since the instrument is computer interfaced, very little technician labor is required to acquire large amounts of information . This sort of precision and cost effectiveness are important attributes of ECIS.
  • ECIS Cultureware™ Disposable Electrode Arrays ECIS Cultureware consists of disposable electrode arrays containing gold film electrodes delineated with an insulating film and mounted on a 20 mil optically clear Lexan* polycarbonate substrate . The well top assembly is made of polystyrene. The gold layer is thin enough to allow microscopic observation of the cells using a standard inverted tissue culture microscope. Each well has a gold electrode at the base, which is 250 µm in diameter and has a surface area for cell attachment and growth of 0.9 cm^2. The well holds a maximum volume of about 500 microliters. The ECIS electrode array is placed in an array holder located in the incubator . The final step in the manufacturing process of the arrays involves exposure of the slides to an oxygen plasma that both cleans the electrodes and sterilizes the chambers and lids. (This step also renders both the substrate and the polystyrene walls of the well very hydrophilic or wettable.)
  •  
  • Each of the 8 wells contains a single circular 250μm diameter active electrode. Each well has a substrate area of 0.8 cm^2 and a maximum volume of 600μL. On average, with a confluent cell layer, approximately 50 to 100 cells will be measured by the electrode, but even a single cell can be observed. Potential uses include: • Cell migration measurements via automated wound healing. • Studying the effects of agents upon cell motions (micromotion) and cell morphology. • Exceptional signal to noise ratio allows studies of very sparse cultures (including single cells). • The single electrode is ideal for correlated microscopy and ECIS experiments. 8W1E
  • Each of the 8 wells contains ten circular 250 μm diameter active electrodes connected in parallel on a common gold pad. Each well has a substrate area of 0.8 cm^2 and a maximum volume of 600 μL. On average, with a confluent cell layer, approximately 500 to 1000 cells will be measured by the electrodes. Potential uses include: • Recording the activities of more cells over a larger region of the substrate. • Studying the effects of agents upon overall morphology changes. • Reducing fluctuations in impedance due to cell micromotion that may obscure subtle changes in impedance. • Cell migration via automated wound healing is normally possible, providing an average of 10 identical wounds. 8W10E
  • Each of the 8 wells has two sets of 20 circular 250 μm diameter active electrodes located on inter-digitated fingers to provide measurements of cells upon a total of 40 electrodes. Each well has a substrate area of 0.8 cm^2 and a maximum volume of 600 μL. On average, with a confluent layer, approximately 2000 to 4000 cells will be measured by the electrodes. The 10E+ arrays are designed to monitor larger numbers of cells, sampling over the entire bottom of the well. Because of the relatively high number of cells, impedance fluctuations due to micromotion are smoothed out and do not obscure subtle changes in impedance due to the experimental conditions. Potential uses include: • Cell-ECM protein interactions. • Signal transduction assays. • Detection of invasion of endothelial cell layers by metastatic cells. • Barrier function measurements. • Cell proliferation. 8W10E+
  • This array is nearly identical to the 8W1E standard array but the active electrodes come in four different diameters (two wells of each size). Diameters are 250, 100, 50 and 25 μm. Since the number of cells monitored depends upon the active electrode area, these special arrays are used to monitor fewer cells with higher sensitivity. Each well has a substrate area of 0.8 cm^2 and a maximum volume of 600 μL. 8W1EDD
  • Each well in this array has two independent single 250 μm diameter active electrodes.The Medusa array is useful for duplicating readings in the same well or to wound/electroporate one electrode while leaving the other as a control within the same well. When connected to the array holder only the upper four wells are measured. To use the other four wells, the array is turned around and the contact pads at the other end are connected. Each well has a substrate area of 0.8 cm^2 and a volume of 600 μL, and with a confluent cell layer, approximately 50-100 cells will cover each electrode. * As the electrode pattern resembles a many headed snake, the name is taken from Greek   mythology and the hair of the infamous Gorgon. 8W2x1E (Medusa*)
  • This array is used to monitor the movement of cells in response to chemical gradients and is the array used in chemotaxis measurements first described by Hadjout, N. et al. (2001)  Biotechniques 31  (5) 1130. The measuring electrode in this array is a thin gold line* between two registry marks. Each well has a substrate area of 0.8 cm^2 and a maximum volume of 600 μL. On average, with a confluent layer, approximately 50 to 100 cells will be monitored by the electrode. *The gold line has the same total area as a 250 μm single circular electrode. ChemoTaxis
  • This is a specialized array with 8 active 250 μm diameter electrodes located in the central region at the base of a flow channel measuring 50 mm long, 5 mm in width and 0.4 mm in height. The flow array is useful for ECIS measurements of cells under perfused conditions or to mimic the shear stress endothelial cells experience in vivo. The channel has an area of 2.5 cm^2 and a channel volume of 100 μL. On average, a confluent layer of approximately 50 to 100 cells will be monitored by each electrode. Flow Array
  • 8W1E Printed Circuit Board (PCB) & 8W10E Printed Circuit Board (PCB) This array is similar to the standard 8W1E and 8W10E but uses a printed circuit board as a base layer. All surfaces are compatible with cell culture as in the standard array. This economy array is opaque and optical cell observations are limited to reflection microscopy (upper chambers can be removed following ECIS measurements). PCB
  •  
  •