Next generation impedance-based cell monitoring with a number of enhanced features added:
- Top model with improved performance
- Handy docking station facilitates hassle-free handling before, during and after the experiment
- Easy readout parameters: TER and Ccl of the cell layer
- Full compatibilty with a broad range of standard cell culture inserts
- Cell Module can be loaded with 24 inserts simultaneously
Like all cellZscope systems, the cellZscope2 is computer-controlled and allows automated fast process monitoring as well as reliable long term measurements over days and weeks. The ohmic resistance (TER, transepithelial / -endothelial resistance) and capacitance (Ccl) of the cell layers under investigation are determined in real-time.
The convincing performance and instrument operations make the cellZscope2 the ideal tool for controlling cellular barriers. In addition it is characterized by an easy maintenance and high user-friendliness. The cellZscope sweeps over a wide frequency range instead of simply measuring at a very few frequency points. This ensures a reliable and unambiguous detection of cell layer properties.
The cellZscope2 is designed to ensure simple operation and maximum flexibility. Its Cell Module provides investigators full apical and basolateral access to all wells. The handy docking station allows the Cell Module to be easily moved from the incubator to laminar flow hood with no cables to unplug.
Single-piece stainless steel “pots” serve as bottom electrodes, as vessels for holding the cell culture medium, and as supports for cell culture inserts.
A magnetic mount makes handling of the upper electrodes comfortable and accurate positioning is guaranteed. The bottom electrodes can be detached easily without the use of tools. These features make the cellZscope systems a snap to clean and sterilize.
Three well sizes are available for use with small (“24-well” type), mid-sized (“12-well” type), or large (“6-well” type) inserts, respectively.
Different sizes of cell culture inserts can be combined within a single Cell Module. Thus allowing the automated monitoring and direct comparison of the barrier properties of cell layers housed in up to three different sized cell culture inserts.
Impedance Based Cell Monitoring – From Short-Term Tight Junction Dynamics To Long-Term Layer Formation
Impedance spectroscopy is the method of choice for analyzing and monitoring cell cultures under physiological conditions. It works label-free, does not require any fixation or staining, and allows to keep the cell cultures which are under investigation alive for subsequent experimental steps. These features combined with a high level of automation in data acquisition and analysis as implemented in the cellZscope® make it the ideal tool for studying epithelial or endothelial cells in vitro. The cellZscope is equally suited for following short-term dynamics in the tight junctions network as well as for long-term monitoring of cellular processes such as layer formation, differentiation, and polarization.
Tight junctions dynamics
The barrier properties of epithelial and endothelial cell layers are determined to a large extend by tight junctions located in the intercellular space. As they form a seal between the apical and basolateral membrane domain they have a strong influence on the paracellular passage of substances. The barrier function is not static but can be deliberately modulated by exposure to specific stimuli. The resulting dynamics of the tight junction network can be conveniently followed by measuring the transepithelial / -endothelial electrical resistance (TER) with the cellZscope. The figure below shows the effect of exposing Madin-Darby canine kidney (MDCK) cells grown confluent on the permeable membranes of standard cell culture inserts to different concentrations of EGTA.
It is well known that this reagent leads to a depletion of extracellular Ca2+ which in turn causes a disassembly of tight junction [1, 2]. The latter is reflected by a significant drop in the TER readings. Subsequent replacement of the EGTA containing medium by standard medium led to a regeneration of the tight junctions network as revealed by increasing TER readings. For validation of the temporary break down of the barrier function two cell cultures were fixed just before removal of EGTA. Samples were then stained for immunofluorescent analysis of cell nuclei and ZO-1 proteins. Imaging by Confocal Laser Scanning Microscopy and comparison with the untreated reference cell culture clearly revealed the disintegration of the tight junctions network induced by EGTA exposure. These findings are in excellent agreement with the TER results and demonstrate the benefits of using a label-free and noninvasive technique as implemented in the cellZscope.
Cell layer formation
Application of impedance spectroscopy for cell analysis is not limited to short-term following of dynamic processes but also allows continuous monitoring of cell cultures for several days or even weeks. The fact that various factors such as seeding density, type of growth medium, concentration of serum, type of substrate coating and temperature have a strong influence on cell layer formation calls for a means to monitor growth continuously while maintaining physiological conditions. Both readout parameters of the cellZscope, the transepithelial / -endothelial electric resistance (TER) and the cell layers’ capacity (Ccl) provide valuable information about the current state of cells as they grow confluent and differentiate. Caco-2 is a well-established cell line derived from human colon adenocarcinoma which is commonly used for drug-transport studies. Precise knowledge of the cell layer’s growth state is mandatory for use as an intestinal permeability model. In the experiment depicted below the cellZscope was employed to follow layer formation and differentiation of Caco-2 cells, with automated data recording beginning right after seeding and continuing for a total time span of more than three weeks.
Except for media exchange at regular time intervals no manual intervention was necessary for recording the data. Thus optimal physiological conditions were maintained throughout the experiment. The full time course reveals distinct differences in the growth behavior of the two different Caco-2 cell lines. Hence, the cellZscope provides quality control for cell layers and allows to have well-defined starting conditions for subsequent experiments such as permeability studies.
nanoAnalytics thanks K. Hardes, V. Heitmann, J. Hüwe, M. Kahns, K. Riehemann (Westfälische Wilhelms-Universität Münster) for their comprehensive support.
 Rutten, M.J., Hoover, R.L., Karnovsky, M.J., Electrical resistance and macromolecular permeability of brain endothelial monolayer cultures, Brain Res. 425, 301 (1987).
 Rothen-Rutishauser, B., Riesen, F.K., Braun, A., Günthert, M., Wunderli-Allenspach, H., Dynamics of Tight and Adherens Junctions Under EGTA Treatment. J. Mem. Biol. 188, 151 (2002).
Monitoring Barrier Properties of MDCK Cell Layers During Depletion of Cell Cholesterol by Methyl-Beta-Cyclodextrin
Plasma membrane lipids such as cholesterol play an important role in regulating the barrier function of epithelial cell layers. Lipids contribute to the structure, assembly and function of tight junctions and thereby have an effect on the selective permeability of the paracellular pathway. A common in vitro approach to study the underlying mechanisms is to selectively alter the lipid composition of the plasma membrane and then employ analytical techniques to determine possible effects on the barrier properties. In this study Madin-Darby canine kidney (MDCK) cells grown on porous membrane inserts were exposed to different concentrations of methyl-ß-cyclodextrin (MBCD). This agent serves as a cholesterol binding reagent and thereby allows one to selectively lower the content of cellular cholesterol.
The cellZscope® provides valuable information on the barrier properties and the differentiation stage of cell layers by measuring their transepithelial electrical resistance (TER) and capacitance (Ccl). In contrast to other analytical techniques these electrical measurements can be performed marker-free on living cells, leaving them fully viable for further experiments. This makes the cellZscope the ideal tool for monitoring cells while they grow confluent and differentiate. Once the cells have reached a steady-state subsequent experiments can be performed while the cellZscope continues to measure the electrical parameters characterizing the barrier function and the stage of differentiation.
Monitoring the cell layer and junction formation
MDCK-II cells were seeded (density 5×105 cells/cm2) in serum-containing medium on the porous polycarbonate membrane (pore density 1×108/cm2, pore size 0.4µm) of standard cell culture inserts (Corning, Transwell®, #3401). Cells were allowed to settle and attach to the membrane for 10 hours. Then the formation of a differentiated cell monolayer with established cell-cell junctions was monitored with the cellZscope by measuring the electrical resistance and capacitance. As shown in the diagram the time course was followed for 60 hours with automatic recording of data points for each of the 24 wells every hour.
Time course of the transepithelial electrical resistance and capacitance of MDCK-II cells. The cellZscope allows continuous data recording while the cell cultures remain in the incubator.
The initial steep increase in TER clearly marks the onset of junction formation. This process was completed after 8 hours when TER reached a maximum with full establishment of a tight junction network. The subsequent decrease in TER can be attributed to an increasing cell number per surface area, i.e. the total perimeter length of cell-cell contacts connected in parallel increases. As a consequence the total resistance of the network, i.e. TER decreases. Finally, after approximately 40 hours TER converged to a steady state level. At this time point the medium was replaced with serum-free medium and the cell layers allowed to adapt for further 20 hours.
Monitoring the barrier function and cell differentiation
The change to serum-free medium was performed in order to minimize cholesterol content in the medium prior to MBCD treatment. Thus MBCD kept its depletion potential for selectively lowering cell cholesterol levels. Then different concentrations of MBCD were added to the top compartment of the wells, i.e. to the apical side of the cell layers. The response of the MDCK cells was analyzed by monitoring TER and Ccl with the cellZscope.
Time courses of TER and Ccl exhibit a dose dependent response of the MDCK-II cells to MBCD treatment (dashed line). Data points represent the averaged mean and error bars the standard deviation of three wells.
The observed initial increase in TER is well know from literature and different interpretations of the underlying biochemical mechanisms were suggested [1, 2]. The results obtained with the cellZscope contribute to ongoing investigations in this field by providing the cell layers’ capacitance Ccl as an independent readout parameter. This type of complemental data recorded simultaneously with TER reveals another dose dependent change in the cell layers’ properties: it directly indicates morphological changes in the plasma membrane.
Results of two independent experiments showing the dose dependent response in TER and Ccl of MDCK-II cells to cholesterol depletion after 30 min. of exposure to MBCD.
The observed reduction in the total membrane capacitance caused by exposure to MBCD is exactly in line with independent investigations based on confocal microscopy : these studies revealed that the depletion of cell cholesterol leads to the retraction of surface microvilli and microridges. Consequently, Ccl shows a significant, dose dependent decrease, since the cell layer’s electrical capacitance directly depends on the morphology of the plasma membrane. In particular, a retraction of protrusions such as microvilli and microridges causes a decrease of the effective surface area and thereby leads to a decrease of the electrical capacitance. The cellZscope is tailored to detect and monitor such changes in the plasma membrane during the full time course of an experiment.
nanoAnalytics thanks K. Hardes and K. Riehemann (Westfälische Wilhelms-Universität Münster) and J. Wegener (Universität Regensburg) for their comprehensive support of this study.
 Stankewich, M., Francis, S.A., Vu, Q.U., Schneeberger, E.E., Lynch, R.D., Alterations in Cell Cholesterol Content Modulate Ca2+-Induced Tight Junction Assembly by MDCK Cells. Lipids 31, 817 (1996).
 Francis, S.A., Kelly, J.M., McCormack, J., Rogers, R.A., Lai, J., Schneeberger, E.E., Lynch, R.D., Rapid reduction of MDCK cell cholesterol by methyl-ß-cyclodextrin alters steady state transepithelial electrical resistance. Eur. J. Cell. Biol. 78, 473 (1999).
 Colarusso, P., Spring, K.R., Reticulated Lipid Probe Fluorescence Reveals MDCK Cell Apical Membrane Topography. Biophys. J. 82, 752 (2002).
Studying Compound Mediated Effects on Primary Cultured Endothelial and Epithelial Cells
Time-resolved monitoring of the transepithelial electrical resistance (TER) of primary cultured endothelial cells derived from porcine brain microvessels, incubated in serum-free medium supplemented with hydrocortisone (orange curve) and without hydrocortisone (blue curve): the experimental data reveal that the TER of the confluent cell layer increases with time in the presence of hydrocortisone. This effect is attributed to a pronounced barrier strengthening of the cerebral endothelial cells.
Time-resolved monitoring of the capacitance (Ccl) of primary cultured epithelial cells derived from porcine choroid plexus, incubated in serum-free medium (orange curve) and in serum-containing medium (blue curve): choroid plexus epithelial cells develop longer and more densely packed microvilli on their apical surface when incubated in a serum-free medium. This differentiation process leads to an increase of the capacitance of the confluent cell layer and can thus be followed noninvasively in situ.
Graphs adapted from Wegener et al., BioTechniques 37, 590 (2004).
Measuring TER — A Comparative Study: cellZscope vs. Handheld Devices with "Chopstick" Electrodes
The cellZscope comes with customized software which provides full instrument control including user defined experiment setup and automated data acquisition. Furthermore, it features comprehensive tools for displaying and statistically analyzing data as well as printing and exporting results.
The user interface is geared to easy operation of the instrument. Main features are:
- Schedule measurements by specifying time intervals for data acquisition according to experimental requirements,
- monitor results in real time,
- enter individual well descriptions and notes,
- log and annotate all experimental events.
The cellZscope software allows to monitor and record all relevant parameters during the course of the experiment, in particular it provides the
- transepithelial / -endothelial electric resistance TER and the
- electric capacitance Ccl
of the cell layer as easy readout parameters.
User-friendly functions for exporting cellZscope data into popular file formats are included, making seminar and manuscript preparations a breeze.
Compatible cell culture inserts
The cellZscope2 is compatible with a variety of standard cell culture inserts from different manufacturers. Three versions of the electrodes are available. They are tailored for use with small (“24-well” type), mid-sized (“12-well” type), or large (“6-well” type) inserts, respectively. Different well sizes can be combined within one Cell Module.
cellZscope is a registered trademark of nanoAnalytics GmbH. Corning, Transwell and Falcon are registered trademarks of Corning Inc. Greiner Bio-One is a registered trademark and ThinCert is a trademark of Greiner Bio-One GmbH. Millipore and Millicell are registered trademarks of Merck KGaA. Nunc is a trademark of Thermo Fisher Scientific Inc. All other brand or product names are trademarks or registered trademarks of their respective holders.
|Controller||26 cm × 6 cm × 27 cm
(10.2 in × 2.4 in × 10.6 in)
|Cell Module||32 cm × 24 cm × 7 cm
(12.6 in × 9.5 in × 2.8 in)
|Station||32 cm × 29 cm × 4 cm
(12.6 in × 11.5 in × 1.6 in)
|Space required in incubator||40 cm × 29 cm × 12 cm
(15.8 in × 11.5 in × 4.8 in)
|Length||150 cm (59 in)|
|Cross section (W×T)||29 mm × 4 mm
(1.2 in × 0.16 in)
|Usually the ribbon cable can be simply fed through the front door of the incubator.|
|Frequency range||1 Hz to 100 kHz|
|Measurement time for 24 wells
- Standard resolution†
- Reduced resolution‡
~ 4.5 min
~ 1.2 min
|Number of wells||24 (4 rows, 6 columns)|
|Power Supply||100 – 240 Vac; 0.8 A; 47 – 63 Hz|
|Minimum requirements for the control computer||- 1.0 GHz processor speed
- 1024 MB RAM
- USB 2.0 port
- 1024×768 display
|Compatible OS||Microsoft Windows® 7/8/10|
|† 1 Hz – 100 kHz, fine frequency resolution
‡ 5 Hz – 100 kHz, coarse frequency resolution