Label-free and time-resolved measurements of cell volume changes by surface plasmon resonance (SPR) spectroscopy

Biosensors and Bioelectronics 25 (2010) 1221–1224

Contents lists available at ScienceDirect

Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios

Short communication

Label-free and time-resolved measurements of cell volume changes by surface plasmon resonance (SPR) spectroscopy R. Robelek ∗ , J. Wegener Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Universitaetsstrasse 31, 93053 Regensburg, Germany

a r t i c l e

i n f o

Article history: Received 15 July 2009 Received in revised form 7 September 2009 Accepted 9 September 2009 Available online 16 September 2009 Keywords: Surface plasmon resonance Biosensor Cell volume change Label-free detection Real-time monitoring

a b s t r a c t Cell volume and its regulation is one of the key players for cellular integrity and a strong indicator for several cell pathologies. But time-resolved volume measurements of adherently grown mammalian cells using established methods, such as extracellular impedance analysis or light microscopy, are complex and time-consuming. In this study, we demonstrate that surface plasmon resonance spectroscopy (SPR) is a powerful transducer device capable of reporting volume changes of cells that are directly grown on the SPR sensor surface. The approach is label-free, non-invasive and provides an outstanding time resolution. In proof-of-principle studies we recorded the volume change of confluent MDCK II cells induced by hypoor hypertonic stimulation in a time-resolved manner. Comparison of the SPR-based experiments reported here with more recent studies using different approaches suggests a direct correlation between SPR signal shift and cell volume changes. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Label-free and time-resolved cell volume measurements of viable cells have been a challenging problem in many areas of experimental and diagnostic biomedical science (Kimelberg et al., 1992). Thus, in the past a variety of techniques have been used to tackle this sensoric task. These include electrophysiological methods, like patch clamping (Satoh et al., 1996) and scanning ion conductance microscopy (Korchev et al., 2000), or methods assessing indirectly the mean cell volume by impedance measurements, like Coulter counting (Grinstein and Furuya, 1984) and volume cytometry (Ateya et al., 2005). These techniques are, however, technically demanding, difficult to apply to more than a small number of samples and are highly dependent on a well-defined and constant conductivity of the experimental buffer with and without osmotic loads. Besides the above mentioned electrochemical techniques, sophisticated optical methods, like interferometry (Farinas et al., 1997), light diffraction (Mcmanus et al., 1993), confocal microscopy (Satoh et al., 1996) and various kinds of three-dimensional fluorescence imaging techniques (Allansson et al., 1999; Crowe et al., 1995), have been used for cell volume measurements. Many of these methodologies require complex microscope configurations and/or laborious imaging and computational procedures. Others involve the introduction of fluorescent labels into the cell, which might ultimately impact viability and permit data acquisition only

for a limited period of time before photobleaching degrades the signal or phototoxicity becomes apparent. Thus, the development of a simple, efficient and non-invasive procedure, which allows recording the time course of cell volume changes, has clear and extensive application potential. Here we report on a biosensor based on surface plasmon resonance (SPR) spectroscopy for the detection of cell volume changes. SPR spectroscopy is an optical method for measuring the refractive index of very thin layers of material adsorbed on a metal sensor layer. A fraction of the incident light energy that hits the surface at a sharply defined angle can interact with the delocalized electrons in the metal film (plasmon) thus reducing the reflected light intensity. The angle of incidence and therefore the SPR signal depends on the refractive index of the adsorbed material (Homola, 2008). In the last years it has been shown that SPR sensors in combination with adherent eukaryotic cells as biorecognition elements can be used to record and investigate cellular processes and morphology changes (Robelek, 2009). As volume changes of cells grown on such an SPR sensor surface always go along with multiple cellular processes that induce a change of the refractive index of the cytoplasm reaching into the evanescent measurement field, SPR should be a very helpful tool for the investigation of cell volume changes. 2. Materials and methods 2.1. Solutions

∗ Corresponding author. Tel.: +49 941 943 4048; fax: +49 941 943 4491. E-mail address: [email protected] (R. Robelek). 0956-5663/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2009.09.016

Experimental buffers were based on Dulbecco’s phosphate buffered saline solution including 0.5 mM MgCl2 ·6H2 O and 1 mM

1222

R. Robelek, J. Wegener / Biosensors and Bioelectronics 25 (2010) 1221–1224

Fig. 1. (a) Phase contrast micrograph of a confluent MDCK II cell layer grown directly on top of a SPR sensor surface. (b) Scheme of the Kretschmann-configured SPR sensor unit for the measurement of cell volume changes.

CaCl2 ·2H2 O (isotonic running buffer, PBS2+ ). To increase the osmolarity of this buffer 100, 200 and 400 mM sucrose (Serva) was added, respectively. Osmolarity was checked using an Osmomat 030 Osmometer (Gonotec, Germany). 2.2. Cell culture Madin-Darby Canine Kidney Strain II (MDCK II) cells were kindly provided by the Institute of Biochemistry, University of Muenster. For stock cultures, the cells were grown to confluence in standard cell culture bottles (25 cm2 , Corning) using Minimal Essential Medium (MEM, 1×) with 1 g/L d-glucose plus 5% (v/v) fetal calf serum, 4 mM l-glutamine and 100 ␮g/mL penicillin/streptomycin. The cultures were kept in an ordinary humidified cell incubator at 37 ◦ C and 5% CO2 . 2.3. SPR sensor preparation 50 nm gold layers thermally evaporated onto high refractive index glass slides were purchased from MiviTec (Germany). The SPR sensor slides were put in small cell culture dishes and were pre-incubated with 3 ml of the standard cell culture medium (see above) for 5 min. The MDCK II cells were removed from the bottom of the cell culture bottles by standard trypsination and suspended in 2.5 ml culture medium. The suspension was diluted 10-fold with culture medium and added to the sensor containing culture dishes. The sensor/cell system was incubated for another 24 h at 37 ◦ C and 5% CO2 . 2.4. SPR measurement SPR measurements were performed with a Biosuplar 6 SPR system (MiviTec, Germany) at room temperature. Data was recorded using the manufacturer’s software and exported to the software Origin6 (OriginLab, USA) for further analysis. 3. Results and discussion 3.1. Setup of SPR sensor To realize the SPR sensor concept for cell volume measurements we cultured MDCK II cells directly on top of gold-coated SPR chips (thickness of gold film 50 nm), which were pre-incubated for 5 min with serum containing culture medium. After obtaining a confluent cell layer, the cells were analyzed by phase contrast microscopy. As can be seen from Fig. 1a the adherent MDCK II layers show the typical cobblestone-like morphology of epithelial cells growing on a standard culture substrate. Staining of the cells with trypan blue

revealed that the cell membranes are intact and impermeable for this vital stain, indicating cell vitality (data not shown). After removing the culture medium and washing the cell layers with PBS2+ , the sensor surfaces were mounted on top of a glass prism using immersion oil matching the refractive index of the glass slide and the prism. The resulting Kretschmann-configured SPR sensor (Fig. 1b) was covered with a polydimethylsiloxane (PDMS) sealed, 2-channel flow cell. The SPR measurements that were performed using the described sensor setup always recorded the signal of both of these channels while they were continuously rinsed with a 10 ␮l/min flow of buffer. On one channel, the so named “measurement channel”, the SPR signal of those MDCK II cells was recorded, that were stimulated to change their cell volume by changing the osmolarity of the measurement buffer, while the “reference channel” showed the signal of MDCK II cells under constant flow of isotonic experimental buffer. By subtracting the reference signal from the SPR signal of the measurement channel the influence of temperature and pressure drifts was eliminated.

3.2. SPR measurement of cell volume changes After obtaining a stable SPR signal from both channels, we changed the buffer of the “measurement channel” from isotonic (270 mOsm/kg) to hypertonic (340–553 mOsm/kg) by adding 100, 200 and 400 mM sucrose to the buffer, respectively. This disaccharide is considered as membrane-impermeable so that its presence in the bathing fluid causes a constant hyperosmotic stimulation of the cells. As shown in one of our previous studies using confocal laser scanning microscopy (Steltenkamp et al., 2006) the cells decrease their volume in response to this stimulus due to osmotic waterflow out of the cells (see micrographs in Supplementary Materials). In this study the cellular volume adaptations to these hypertonic stimulations were followed online by the SPR signal. An inverse experiment was performed by applying a hypotonic stimulation, which results in a cell volume increase. Here the isotonic buffer was diluted by the addition of water yielding an osmolarity of 135 mOsmol/kg. For all measurements the “reference channel” was continuously rinsed with the original isotonic buffer. Fig. 2a shows a selection of the reference corrected SPR sensograms that were obtained during the hyper- and hypotonic stimulations of the MDCK II layer. For the hypertonic stimulations we observed a rapid increase of the SPR signal immediately after the cell layer was rinsed with the hypertonic medium. The increase slowed down and reached a new, stable equilibrium level after about 6 min. By changing the osmolarity of the buffer back to the original isotonic value of 270 mOsmol/kg the SPR signal returns to the starting level. We postulate that the SPR signal correlates to the cellular volume change, as the loss of cellular water from the cytosol causes

R. Robelek, J. Wegener / Biosensors and Bioelectronics 25 (2010) 1221–1224

1223

Fig. 2. (a) SPR curves obtained during hyper- and hypotonic stimulation, respectively, of the sensor adherent MDCK II cell layers (T = 25 ◦ C). Starting from isotonic conditions (270 mOsmol/kg) the cell layer was rinsed with a () 340, (×) 410 and () 135 mOsmol/kg experimental buffer (starting point is indicated by black arrow). The grey arrow indicates the change back towards the isotonic running buffer. (b) Plot of relative SPR signal changes as a function of relative osmolarity differences in the measurement buffer. The data points were linearly fitted resulting in a correlation coefficient of R = 0.991. (c) Curve fitting of SPR curve recorded during hypertonic stimulation (Osm = +70 mOsmol/kg) using a mono-exponential function.

an increased cytoplasmic concentration of intracellular osmolytes and, thus, an increased refractive index close to the sensor surface. Supposedly only that fraction of the cell volume that resides within the evanescent field of the SPR close to the substrate-facing membrane contributes to the recorded signal. Furthermore from these hypertonic stimulation experiments we gained some other strong hints, that the measured SPR signal corresponds to the cell volume change: (i) as shown in Fig. 2b, the relative SPR signal shift between the starting (isotonic) and the final signal level (hypertonic or hypotonic) increases linearly with the relative change in buffer osmolarity (correlation coefficient R = 0.991). This result is in good agreement with previously published studies, reporting that MDCK cells show a volume decrease that is linearly dependent on the osmolarity of the stimulation buffer (Roy and Sauve, 1987). (ii) Following the data analysis procedures of other groups (Heo et al., 2008; Maric et al., 2001) we fitted the increase of the SPR signal after stimulating with a 340 mOsm/kg buffer using a mono-exponential function (Fig. 2c). The time constant  that could be extracted from such curve fitting amounts to 1.45 ± 0.12 min, which is perfectly inline with the range of published time constants for a cellular volume change of kidney epithelium cells when they are osmotically challenged. Consistent with the explanation of an increasing SPR signal after hypertonic stimulation, an exchange of the isotonic against

a hypotonic buffer (135 mOsmol/kg) causes an overall decrease of the SPR signal (Fig. 2a, triangles). Water flowing from the extracellular space into the cell, dilutes the cytosol, which causes the refractive index within the evanescent field, and thus the SPR signal, to decrease. But in contrast to the monotone cellular reaction following a hypertonic stimulation, the MDCK II layer shows a more complex behaviour when challenged by hypotonic conditions. Especially in the starting phase of the stimulation the SPR signal begins to increase for a short period of time before it shows a prolonged decrease. In addition the signal decrease and thus the cellular reaction triggered by the hypotonic stimulation is not as fast as expected. This points to a second cellular reaction that overlays a straight cell volume increase. MDCK cells were reported to be able to perform a regulatory volume decrease (RVD) when stimulated with hypotonic medium but do not show any regulatory volume increase (RVI) when contacted with hypertonic medium (Roy and Sauve, 1987). The observed differences in the SPR signals for the two non-isotonic stimulation cases might refer exactly to this fact. Even if a resilient correlation between RVD and the complex SPR signal shift in the hypotonic stimulation case will have to be proven by further experiments, the overall time course of the SPR signal resembles strongly published data of MDCK cell behaviour when challenged with hypotonic conditions (Roy and Sauve, 1987). We consider this observation as

1224

R. Robelek, J. Wegener / Biosensors and Bioelectronics 25 (2010) 1221–1224

the sensor surface induced by the non-isotonic extracellular environment. 4. Conclusions

Fig. 3. SPR curves recorded during exposure of SPR sensors either cell free () or covered by a confluent monolayer of MDCK-II cells (×) with isotonic running buffer containing 10% (w/v) PVP (T = 25 ◦ C). Time point for buffer change from isotonic running buffer (PBS2+ ) to PBS2+ + PVP is indicated by a black arrow. The grey arrow indicates the change back to the isotonic running buffer.

another indication, that the SPR signal directly mirrors cell volume changes. 3.3. Preclusion of bulk refractive index influences To proof that the measured SPR signal reflects a cellular reaction to an extracellular non-isotonic challenge and not a simple change in the bulk refractive index of the incubation buffer, we compared the SPR signal for a cell-covered SPR sensor with a cell free sensor surface when both surfaces were rinsed with a medium that raises the bulk refractive index substantially without affecting the buffer osmolarity. For this purpose we added 10% (w/v) polyvinylpyrolidone (PVP, average MW = 360 kDa) to the standard isotonic running buffer (PBS2+ ) to shift its refractive index by Rf = 0.02, a value that would cause a huge bulk refractive index change. Because of the high molecular mass of the PVP, the osmolarity of the rinsing buffer, however, is only changed to a negligible extent (284 mOsmol/kg). Fig. 3 shows the sensograms that were recorded in this experiment. The cell free sensor surface, that was pre-treated with culture medium and standard rinsing buffer in the same way as the cellcovered surface, shows a massive signal shift, when contacted with the PVP containing buffer. The signal shape is typical for a bulk refractive index change with a very rapid increase and decrease phase. In contrast the cell-covered SPR sensor surface exhibits only a minor, very prolonged signal change. This result proofs, that the confluent MDCK II cell monolayer, that shows an average cell height of 6 ␮m (Kersting et al., 1993), shields the evanescent field (average height 250 nm) from a contact with the PVP containing buffer, and so prevents a change in the SPR signal due to bulk refractive index changes. Thus, the only explanation for the observed SPR signal shifts for cell-covered SPR sensors in all the above mentioned experiments is a change of the intracellular refractive index close to

SPR-based measurements of volume changes in adherently grown mammalian cells are easy to perform, label-free, noninvasive and provide a time resolution that can be improved down to the millisecond regime. It has already been shown with other whole-cell biosensor devices that gold-coated surfaces, as they are used in the SPR approach, are inert and fully cytocompatible growth surfaces that allow for a huge variety of chemical surface modifications if needed. In principle, the SPR approach described here can be applied to all adherently growing mammalian cell types to study a variety of biomedical scenarios that are associated with cell volume changes. The proof-of-principle experiments reported above might pave the way for label-free cell volume measurements with spatial resolution when imaging SPR devices are applied. Moreover, the SPR approach might be extended to a platform technology to screen for therapeutic modulators of cell volume changes. Acknowledgements The authors would like to acknowledge the expert help of Dr. Thomas Hirsch in setting up the SPR device. Generous financial support by the Kurt-Eberhard Bode Stiftung is gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2009.09.016. References Allansson, L., Khatibi, S., Gustavsson, T., Blomstrand, F., Olsson, T., Hansson, E., 1999. Journal of Neuroscience Methods 93 (1), 1–11. Ateya, D.A., Sachs, F., Gottlieb, P.A., Besch, S., Hua, S.Z., 2005. Analytical Chemistry 77 (5), 1290–1294. Crowe, W.E., Altamirano, J., Huerto, L., Alvarezleefmans, F.J., 1995. Neuroscience 69 (1), 283–296. Farinas, J., Kneen, M., Moore, M., Verkman, A.S., 1997. Journal of General Physiology 110 (3), 283–296. Grinstein, S., Furuya, W., 1984. Biochemical and Biophysical Research Communications 122 (2), 755–762. Heo, J., Meng, F., Hua, S.Z., 2008. Analytical Chemistry 80 (18), 6974–6980. Homola, J., 2008. Chemical Reviews 108 (2), 462–493. Kersting, U., Schwab, A., Treidtel, M., Pfaller, W., Gstraunthaler, G., Steigner, W., Oberleithner, H., 1993. Cellular Physiology and Biochemistry 3 (1), 42–55. Kimelberg, H.K., Oconnor, E.R., Sankar, P., Keese, C., 1992. Canadian Journal of Physiology and Pharmacology 70, S323–S333. Korchev, Y.E., Gorelik, J., Lab, M.J., Sviderskaya, E.V., Johnston, C.L., Coombes, C.R., Vodyanoy, I., Edwards, C.R.W., 2000. Biophysical Journal 78 (1), 451–457. Maric, K., Wiesner, B., Lorenz, D., Klussmann, E., Betz, T., Rosenthal, W., 2001. Biophysical Journal 80 (4), 1783–1790. Mcmanus, M., Fischbarg, J., Sun, A., Hebert, S., Strange, K., 1993. American Journal of Physiology 265 (2), C562–C570. Robelek, R., 2009. Bioanalytical Reviews, in press. Roy, G., Sauve, R., 1987. Journal of Membrane Biology 100 (1), 83–96. Satoh, H., Delbridge, L.M.D., Blatter, L.A., Bers, D.M., 1996. Biophysical Journal 70 (3), 1494–1504. Steltenkamp, S., Rommel, C., Wegener, J., Janshoff, A., 2006. Small 2 (8–9), 1016–1020.