Simultaneous Impedance Spectroscopy and Optical Microscopy to Investigate Cells Attachment and Spreading

Tuesday, October 13, 2015: 11:40
Borein B (Hyatt Regency)


In a normal environment cells are responding to the signals coming from their external environment, and the way they react often determines their fate. These signals can come from multiple soluble chemicals (soluble cues) such as secreted proteins from neighboring cells. These cues usually trigger incredibly complicated pathway of events inside the cells, which lead to certain cellular responses. The mechanisms behind the responses are even more sophisticated as physical property of surrounding environment (adhesive cues) such as ligands in extracellular matrix (ECM) have also their own effect on the signaling pathways and the overall cells responses. Specifically, the Arg-Gly-Asp ligands present in ECM have been mainly known for determining the extent of the cells attachment and spreading in its environment through binding to cellular integrins. It is crucial to achieve a better understanding of the possible links between signaling networks provoked by soluble and adhesive cues. We have coupled fluorescence microscopy and electrochemical Impedance spectroscopy simultaneously to provide more comprehensive information about this connection. To model the adhesive cues, the interdigitated Indium−tin oxide (ITO) surfaces were modified to present different RGD densities. Fluorescence microscopy was used to monitor the activity of G-protein coupled receptors (GPCR) by measuring the intracellular calcium flow, while the impedance spectroscopy was used to track the minute morphological changes all in response to histamine, as an examples of soluble cues, addition on the modified ITO surfaces.

The ITO surfaces with different RGD ligand spacing were prepared by modifying the surfaces with different ratios of hydroxyl-terminated and methoxy-terminated 1-aminohexa(ethylene oxide), followed by incubation in 20 µg/ml RGD solution on a cell-inert background, which was developed on the surface previously. The cell-surface interaction was investigated by seeding 104 HeLa cells/ cm2 on surfaces for 3 h, following by fixing, and staining with phalloidine-Alexa 488 to determine the extent of cells spreading. For impedance spectroscopy, the resistance of the cell layer was measured at 40 kHz, since plating the cells, while the calcium flow in response to addition of histamine was measured using fluorescence microscopy after loading the cells with Fura-2 AM calcium dye. A custom-made chamber from Micrux (Oviedo, Spain) was used to run the simultaneous experiment.

The fixed cell-surface study showed that the cells morphology and spreading is highly dependent to the amount of RGD ligands present on surfaces. The microscopy results indicates that histamine mediated GPCRs activity can significantly induce the flow of calcium in cytosol. In contrast, the impedance spectroscopy showed that altering the ECM-cell integrin interaction by introducing different RGD ligands to the surface modulates the cells morphology on the surface. More importantly, we observed that the presence of different RGD ligands on the surfaces dramatically changed the amount of released calcium as Figure 1 shows. Surfaces modified with 1:103 of hydroxyl-terminated and methoxy-terminated 1-aminohexa(ethylene oxide) was demonstrating the highest amount of released intracellular calcium, while it showed the lowest increase in the amount of impedance among the surfaces that were used in this experiment. 

Cells response to histamine molecules, as soluble cues, trough GPCR was investigated on interdigitated ITO surfaces modified with different RGD ligand densities which acts as cells adhesive cues. The results indicate strong correlation between the effect of soluble cues and adhesive cues. Understanding this highly interconnected signaling networks is vital to biomedical engineering science.  

Figure 1. The percentage of increase in the ratiometric value of Fura-2 AM in response to 100 µM histamine on ITO surfaces modified with different ratio of 1-aminohexa(ethylene oxide) to 1- aminohexa(ethylene oxide) monomethyl ether. The image is an example of the fluorescence images showing the increase in calcium flow in response to histamine, where blow-red refers to low-high.