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 (C_cl) 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.

<sub>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).</sub>

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 (C_cl). 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 C_cl with the cellZscope.

Time courses of TER and C_cl 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 C_cl 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 C_cl 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, C_cl 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.

<sub>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).</sub>

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 (C_cl) 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).}