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Adhesive Cell Growth and Microscopy on O2 and pH Sensor Foils
Impact of coating on the adhesion of cells upon VisiSens™ sensor foils SF-RPSu4 and SF-HP5R
C. Schmittlein1, R. J. Meier2, G. Liebsch2, J. Wegener1,3
1Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Germany
2PreSens Precision Sensing GmbH, Regensburg, Germany
3Fraunhofer Research Institution for Microsystems and Solid State Technologies (EMFT), Regensburg, Germany
Suspension of Madin-Darby Canine Kidney cells (MDCK-II) were plated on VisiSens™ sensor foils SF-RPSu4 (O2) and SF-HP5R (pH) that were integrated in regular Petri dishes. Cell adhesion on a regular Petri dish surface, non-coated sensor foils and sensor foils coated with different adhesion promoting substances was determined by staining and microscopic analysis. After the initial settings were established normal rat kidney cells (NRK), bovine aortic endothelial cells (BAEC) and human glioblastoma cells (U-373 MG) were included in this study. We found that the sensor foils do not have any toxic effect on the different cell lines and that the cells grow well on the sensor foils with no significant difference to regular cell culture substrates.
Growing animal cells directly on the surface of an O2 or pH sensor foil in combination with fluorescence imaging allows quantifying the concentration of both analytes in the direct microenvironment of the adherent cells instead of measuring their concentration with a cell-free but distant sensor in the bulk. The short distance between sensor foil and cell of only 50 to 200 nm provides local readings of oxygen and pH concentration at the cell surface with high temporal and spatial resolution. It avoids the inherent latency in response when an integrating sensor is placed several cell diameters away which is often very position-dependent and additionally affected by more complex diffusion and convection of the analyte. This study addresses the impact of sensor surface coating with adhesive substances on the cell adhesion process and subsequent sensor performance.
Materials & Methods
MDCK II and NRK cells (Leibniz Institute DSMZ, Germany), BAEC (provided by Dr. Zink and Prof. Rösen from the Diabetes-Froschungsinstitut Düsseldorf, Germany) and U-373 MG cells (provided by Prof. Buschauer, Institute of Pharmacy, University of Regensburg, Germany) were seeded in sub-confluent (5.0 x 104 cells cm-2) or confluent (4.5 x 105 cells cm-2) density on the sensor films.
The imaging sensor foils SF-RPSu4 and SF-HP5R (PreSens) were used as cell culture substrate. The SF-RPSu4 sensor foils consist of a reddish oxygen sensitive layer on a transparent support foil and a white optical isolation layer (OIW) on top, which can easily be removed, by peeling it off (Fig. 1). The O2 sensitive layer has different surface properties compared to the optical isolation layer, so both sensors with and without the OIW were tested as cell substrates. pH sensor foils come with a non-removable OIW layer, so that only one type of foil (with OIW) was tested here. The sensor foils were immobilized on the bottom of standard 3.5 cm diameter Petri dishes (Saerstedt) using a biocompatible silicone adhesive (SG-1). The functionalized dishes were exposed to violet Ar plasma for 1 min to clean and sterilize prior to use. Fetal calf serum (FCS), fibronectin, gelatin and poly-L-lysine were used as adhesion promoting coatings and applied to the sensor foils as listed in Table 1.
24 hours after seeding cells on O2 sensor foils were treated with a vital stain consisting of 2 µM Calcein-AM (Invitorgen, Ex 488 nm, Em 530 nm) and 4 µM Ethidium-Homo-Dimer (Biotrend Chemikalien, Ex 528 nm, Em 617 nm) in PBS++ (37 °C, 30 min).
Since the characteristics of the pH sensor foils interfere with the live/dead staining applied before, the cells seeded on pH sensitive foils were stained by TRITC-phalloidin (Invitrogen, Ex 515 nm, Em 650 nm) to document the architecture of their actin cytoskeleton. As both types of sensor foils are non-transparent, cells were visualized using an upright confocal laser scanning microscope (Nikon Eclipse90i) using a 10x magnification.
Cell Adhesion on O2 Sensor Foils SF-RPSu4-OIW
O2 sensor foils with the optical isolation layer (SF-RPSu4) were coated with different adhesion promoting materials (see Tab. 1) to investigate the coating's individual impact on cell adhesion. Cell adhesion upon uncoated sensor foils (Fig. 2 red box) and standard cell culture Petri dishes were used as reference. Figure 2A shows sub-confluent MDCK II cells after live/dead staining by CAM/EtHD. The green cytoplasmic fluorescence of CAM indicates that cells are vital on all substrates with only a few red fluorescing nuclei of dead cells. However on both, coated and non-coated sensor foils, a fraction of cells exhibit only weak adhesion to the substrate, adopting a rounded morphology. The number of fully spread cells is lower on treated and un-treated sensor foils compared to control conditions. The number of dead cells marked by the red nuclear emission of EtHD is not increased when cells are cultivated on sensor foils indicating that the foils are not toxic, but only less adhesive.
Micrographs grouped in Figure 2B show the results for complete cell layers established 24 h after seeding. The vital stain of cells cultured on non-coated, fibronectin and gelatin coated sensor foils indicates vital cells and a similar cell coverage compared to the control. However, the number of dead cells observed on non-coated foils is increased by about 50 % compared to the reference and the coated sensor foils. Furthermore, non-coated sensor foils lack a confluent cell layer which is not yet fully established as on the coated substrate.
Cell Adhesion on O2 Sensor Foils SF-RPSu4 without OIW
MDCK II cells can be cultured directly on the surface of the sensor foils' O2 sensitive layer as demonstrated in Figure 3. The results show no significant difference between control, coated or non-coated sensor foils. The additional coating did provide neither an improved adhesion nor a significantly different number of dead cells on any of the substrates in particular when the cells were allowed to grow to confluence.
Additionally, three other cell lines were analyzed. Micrographs of BAEC, NRK and U-373 cells exposed to CAM and EtHD are grouped in Figure 4. Similar to the results obtained for MDCK II cells the other cell lines do also fully adhere and spread upon non-coated oxygen sensor foils without the optical isolation layer. There was no significant difference in cell adhesion compared to standard cell culture dishes when cells had been grown to confluence. However, a minor increase in the number of dead, rounded-up cells is observed when cells are cultured on coated or non-coated oxygen sensor foils. Accordingly, coating of sensor foils with adhesion promoting materials does not provide any significant improvement in adhesion or viability of the cells under study.
Cell Adhesion on pH Sensor Foils SF-HP5R
pH sensor foils SF-HP5R were also tested as culture substrate with or without the adhesion promoting coatings that have been used for the O2 sensor foils before. Cells were again studied as sub-confluent or confluent cell monolayers. The fluorescence micrographs show that cells are attached to the surface and express a well-established actin cytoskeleton with its characteristic features like the very prominent actin belt. No significant difference between control, non-coated and coated sensor foils was observed. Thus, pH sensor foils can be used without prior coating with adhesion promoting materials.
The sensor foils SF-RPSu4 and SF-HP5R were tested for their biocompatibility in general and their ability to promote cell adhesion in particular. When the standard SF-RPSu4 oxygen sensor foils with OIW layer for the VisiSens Imaging System are used as culture substrates, coating of the sensor foils with an adhesion promoting compound like gelatin or fibronectin is recommended to facilitate cell attachment and spreading upon the sensor foil. When the OIW is removed from the O2 sensitive layer of SF-RPSu4, coating is not mandatory as demonstrated in experiments using MDCK II, NRK, BAEC or U-373 cells. The pH sensor foils SF-HP5R are compatible with MDCK II cell adhesion and spreading. Since other cell lines than those included in this study may attach and spread differently, we recommend using serum, culture media containing serum or proteins like fibronectin or poly-L-lysine for coating and improved adhesion.