cellZscope+

cellZscope 2

Das Highend-Model zur Messung der Impedanz von barrierebildenden Zellkulturen. Eigenschaften und exklusive Features sind:

  • Topmodell mit verbesserter Messleistung
  • Praktische Dockingstation erlaubt einfaches Handling vor, während und nach dem Experiment
  • Automatische Bestimmung von TER und Ccl der Zellschicht
  • Volle Kompatibilität mit einer Vielzahl von Standard-Zellkultureinsätzen von diversen Herstellern
  • Bestückbar mit bis zu 24 Einsätzen gleichzeitig

Details

Das cellZscope2 wird über einen Computer angesteuert und erlaubt sowohl das Verfolgen von schnellen Prozessen als auch Langzeitmessungen über Tage und sogar Wochen. Der ohmsche Widerstand (TER, transepithelial / -endothelial resistance) und die Kapazität (Ccl) der jeweiligen Zellschicht in Echtzeit bestimmt und ausgegeben.

Komfortable Bedienung und leichte Wartung sind beim cellZscope2 garantiert und machen es zum idealen Instrument um Zellbarrieren zu überwachen. Gemessen wird über ein breites Frequenzspektrum anstelle von Einzelmessungen bei wenigen Einzelfrequenzen. Hierdurch wird eine zuverlässige und eindeutige Bestimmung der Zellschichteigenschaften gewährleistet.

cellZscope - Hardware

Das cellZscope2 ist auf eine besonders einfache Handhabung und maximale Flexibilität ausgelegt. Sowohl der apikale als auch der basolaterale Bereich der einzelnen Wells sind zugänglich. Durch die praktische Dockingstation lässt sich das Zellmodul für Mediumwechsel und Behandlung besonders leicht zur sterilen Werkbank transportieren.

cellZscope - Hardware

Solide "Töpfchen" aus Edelstahl dienen als Bodenelektroden, als Gefäße zur Aufnahme des Zellkulturmediums und als Träger für die Zellkultureinsätze.

cellZscope - Hardware

Die oberen Elektroden sind magnetisch am Deckel befestigt. Hierdurch sind leichtes Handling und präzise Positionierung gewährleistet. Obere wie untere Elektroden können einfach und ohne dass Werkzeug notwendig ist demontiert werden. Hierdurch ist es mühelos möglich die Wells zu reinigen und steril zu halten.

cellZscope - Hardware

Das Zellmodul kann mit bis zu 24 Töpfchen, die jeweils für die Verwendung von kleinen ("24-well"-Typ), mittelgroßen ("12-well"-Typ) bzw. großen Zellkultureinsätzen ("6-well"-Typ) optimiert sind, bestückt werden.

Innerhalb eines Zellmoduls können verschieden große Zellkultureinsätze kombiniert werden. Dies erlaubt prinzipiell das parallele Beobachten und Vergleichen der Barriereeigenschaften von Zellschichten in bis zu drei unterschiedlich großen Zellkultureinsätzen.


Anwendungen

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.

further details...

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.

cellZscope - MDCKII cells

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.

cellZscope - Caco2 cells

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.

[1] Rutten, M.J., Hoover, R.L., Karnovsky, M.J., Electrical resistance and macromolecular permeability of brain endothelial monolayer cultures, Brain Res. 425, 301 (1987).
[2] 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.

further details...

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.

 

cellZscope - MDCKII cells

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.

 

cellZscope - MDCKII cells

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.

 

cellZscope - MDCKII cells

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 [3]: 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.

[1] 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).
[2] 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).
[3] 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

further details...

 

 

cellZscope - primary cultured endothelial cells derived from porcine brain microvessels

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.

 

 

cellZscope - primary cultured epithelial cells derived from porcine choroid plexus

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

 


Software

Das cellZscope wird inklusive einer maßgeschneiderten Software ausgeliefert. Mittels dieser werden die Daten erfasst und die Messprozedur kann gesteuert und modifiziert werden. Darüber hinaus bietet sie umfassende Möglichkeiten zur statistischen Analyse, grafischen Ausgabe oder Export der Messwerte.

cellZscope - Software

Die Benutzeroberfläche ist intuitiv und ermöglicht eine einfache Bedienung des Gerätes. Hauptmerkmale sind:

  • Messintervalle können entsprechend der experimentellen Anforderungen geplant und definiert werden,
  • Messergebnisse lassen sich in Echtzeit anzeigen,
  • umfassende Möglichkeiten zur Protokollierung und Beschreibung des Versuches,
  • automatische Aufzeichnung von Messereignissen.
cellZscope - Software

Die cellZscope Software ermöglicht es, alle relevanten Parameter im Verlauf des Experiments zu beobachten und aufzuzeichnen, insbesondere sind dies der

  • transepitheliale / -endotheliale elektrische Widerstand TER und die
  • elektrische Kapazität Ccl

der Zellschicht.

cellZscope - Software

Die Messdaten können problemlos in gängigen Formaten exportiert und für Manuskripte oder Vorträge weiter verarbeitet werden.

Kompatible Zellkultureinsätze

cellZscope - Well-Formate

Das cellZscope2 ist kompatibel mit einer Vielzahl von Standard-Zellkultureinsätzen verschiedener Hersteller. Drei Versionen der Elektroden sind verfügbar. Sie sind für die Verwendung mit kleinen ("24-Well" -Typ), mittleren ("12-Well" -Typ) bzw. großen ("6-Well" -Typ) Einsätzen vorgesehen. Unterschiedliche Töpfchengrößen können in einem Zellmodul kombiniert werden.

cellZscope - Kompatible Zellkultureinsätze
Beispiele einiger kompatibler Zellkultureinsätze (nanoAnalytics GmbH übernimmt keine Verantwortung oder Haftung für fehlerhafte oder ungenaue Angaben).


Corning, Transwell und Falcon sind eingetragene Marken von Corning Inc. Greiner Bio-One ist eine eingetragene Marke und ThinCert eine Marke der Greiner Bio-One GmbH. Millipore und Millicell sind eingetragene Marken der Merck KGaA. Nunc ist eine eingetragene Marke von Thermo Fisher Scientific Inc. Alle anderen Marken- oder Produktnamen sind Marken oder eingetragene Marken ihrer jeweiligen Inhaber.

Spezifikationen

Maße (L×B×H)

Controller 26 cm × 6 cm × 27 cm
(10.2 in × 2.4 in × 10.6 in)
Zellmodul 32 cm × 24 cm × 7 cm
(12.6 in × 9.5 in × 2.8 in)
Station 32 cm × 24 cm × 4 cm
(12.6 in × 9.5 in × 1.6 in)
Platzbedarf im Inkubator 40 cm × 29 cm × 13 cm
(15.8 in × 11.5 in × 5.1 in)

Verbindungskabel

Länge 150 cm (59 in)
Querschnitt (B×D) 29 mm × 4 mm
(1.2 in × 0.16 in)
Üblicherweise kann das Flachbandkabel einfach durch die Vordertür des Inkubators geführt werden.

Messparameter

Frequenzbereich 1 Hz to 100 kHz
Messdauer für 24 wells
- Standardauflösung
- Reduzierte Auflösung
 
~ 4.5 Min
~ 1.2 Min

Zellmodul

Anzahl Wells 24 (4 Reihen á 6 Spalten)
6-well Format
12-well Format
24-well Format

Sonstiges

Netzteil 100 – 240 Vac; 0.8 A; 47 – 63 Hz
Mindestanforderungen an den Computer - 1.0 GHz Prozessorgeschwindigkeit
- 1024 MB RAM
- USB 2.0 Anschluss
- 1024×768 Display
Kompatible Betriebssysteme Microsoft Windows® 7/8/10
1 Hz – 100 kHz, feine Frequenzauflösung
5 Hz – 100 kHz, grobe Frequenzauflösung