ECIS Based Electric Fence Method for Measurement of Human Keratinocyte Migration on Different Substrates.

Electric Cell-substrate Impedance Sensing (ECIS) is an impedance-based, real-time, and label-free measuring system for monitoring cellular activities in tissue culture. Previously, ECIS wound healing assay has been used to wound cells with high electric current and monitor the subsequent cell migration. In this study, we applied ECIS electric fence (EF) method, an alternative to electrical wounding, to assess the effects of different surface coatings on human keratinocyte (HaCaT) migration. The EF prevents inoculated cells from attaching or migrating to the fenced electrode surface while maintaining the integrity of the surface coating. After the EF is turned off, cells migrate into the cell-free area, and the increase in measured impedance is monitored. We cultured HaCaT cells on gold electrodes without coating or coated with poly-L-lysin (PLL), poly-D-lysine (PDL), or type-I collagen. We quantified migration rates according to the different slopes in the impedance time series. It was observed that either poly-L-lysine (PLL) or poly-D-lysine (PDL) limits cell adhesion and migration rates. Furthermore, the surface charge of the coated substrate in the culture condition positively correlates with the cell adhesion and migration process. Our results indicate that the EF method is useful for determining cell migration rates on specific surface coatings.

Detecting Effects of Low Levels of FCCP on Stem Cell Micromotion and Wound-Healing Migration by Time-Series Capacitance Measurement.

Electric cell-substrate impedance sensing (ECIS) has been used as a real-time impedance-based method to quantify cell behavior in tissue culture. The method is capable of measuring both the resistance and capacitance of a cell-covered microelectrode at various AC frequencies. In this study, we demonstrate the application of high-frequency capacitance measurement (f = 40 or 64 kHz) for the sensitive detection of both the micromotion and wound-healing migration of human mesenchymal stem cells (hMSCs). Impedance measurements of cell-covered electrodes upon the challenge of various concentrations of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), from 0.1 to 30 µM, were conducted using ECIS. FCCP is an uncoupler of mitochondrial oxidative phosphorylation (OXPHOS), thereby reducing mitochondrial ATP production. By numerically analyzing the time-series capacitance data, a dose-dependent decrease in hMSC micromotion and wound-healing migration was observed, and the effect was significantly detected at levels as low as 0.1 µM. While most reported works with ECIS use the resistance/impedance time series, our results suggest the potential use of high-frequency capacitance time series for assessing migratory cell behavior such as micromotion and wound-healing migration.

Use of Discrete Wavelet Transform to Assess Impedance Fluctuations Obtained from Cellular Micromotion.

Electric cell-substrate impedance sensing (ECIS) is an attractive method for monitoring cell behaviors in tissue culture in real time. The time series impedance fluctuations of the cell-covered electrodes measured by ECIS are the phenomena accompanying cellular micromotion as cells continually rearrange their cell-cell and cell-substrate adhesion sites. Accurate assessment of these fluctuations to extract useful information from raw data is important for both scientific and practical purposes. In this study, we apply discrete wavelet transform (DWT) to analyze the concentration-dependent effect of cytochalasin B on human umbilical vein endothelial cells (HUVECs). The sampling rate of the impedance time series is 1 Hz and each data set consists of 2048 points. Our results demonstrate that, in the Daubechies (db) wavelet family, db1 is the optimal mother wavelet function for DWT-based analysis to assess the effect of cytochalasin B on HUVEC micromotion. By calculating the energy, standard deviation, variance, and signal magnitude area of DWT detail coefficients at level 1, we are able to significantly distinguish cytotoxic concentrations of cytochalasin B as low as 0.1 µM, and in a concentration-dependent manner. Furthermore, DWT-based analysis indicates the possibility to decrease the sampling rate of the micromotion measurement from 1 Hz to 1/16 Hz without decreasing the discerning power. The statistical measures of DWT detail coefficients are effective methods for determining both the sampling rate and the number of individual samples for ECIS-based micromotion assays.

Electrochemical Assessment of Anticancer Compounds on the Human Tongue Squamous Carcinoma Cells.

The most common oral cancer is squamous cell carcinoma (SCC) and its highest occurrence is in the tongue. Almost 30% of patients with one primary head and neck tumor will have a second primary malignancy. In recent studies, two novel plant extracts, andrographolide and cannabidiol (CBD), have been exploited for their anticancer effects. Here, we investigated the cytotoxic effects of these two compounds on SCC-25 cells, a human tongue squamous carcinoma cell line, and compared the outcomes with two chemotherapeutic drugs, cisplatin and fluorouracil. Electric cell substrate impedance sensing (ECIS) system was applied to measure frequency- and time-dependent impedance of SCC-25 cell-covered electrodes and to further assess subtle changes in cell morphology and micromotion in response to different concentrations (0, 10, 30, 100, and 300 µM) of these compounds. AlamarBlue and Annexin V/7-AAD binding assays were used to measure the concentration dependent changes in viability and apoptosis of SCC-25 cells. Our results demonstrate that 24 hours after exposure to 30 µM CBD can significantly decrease the micromotion rate, damage the integrity of cell morphology, reduce cell viability, and induce higher apoptosis in treated SCC-25 cells, while the other three drugs attain similar effects at the concentration of 100 µM or higher. The apoptosis-induced changes in cell morphology and micromotion monitored by ECIS correlate well with biochemical assays. Thus, both frequency- and time-dependent impedance measurements using ECIS can be used to real-time follow cancer cell activities in response to anticancer drugs with different temporal cytotoxicity profiles.

Application of ECIS to Assess FCCP-Induced Changes of MSC Micromotion and Wound Healing Migration.

Electric cell-substrate impedance sensing (ECIS) is an emerging technique for sensitively monitoring morphological changes of adherent cells in tissue culture. In this study, human mesenchymal stem cells (hMSCs) were exposed to different concentrations of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) for 20 h and their subsequent concentration-dependent responses in micromotion and wound healing migration were measured by ECIS. FCCP disrupts ATP synthesis and results in a decrease in cell migration rates. To detect the change of cell micromotion in response to FCCP challenge, time-series resistances of cell-covered electrodes were monitored and the values of variance were calculated to verify the difference. While Seahorse XF-24 extracellular flux analyzer can detect the effect of FCCP at 3 µM concentration, the variance calculation of the time-series resistances measured at 4 kHz can detect the effect of FCCP at concentrations as low as 1 µM. For wound healing migration, the recovery resistance curves were fitted by sigmoid curve and the hill slope showed a concentration-dependent decline from 0.3 µM to 3 µM, indicating a decrease in cell migration rate. Moreover, dose dependent incline of the inflection points from 0.3 µM to 3 µM FCCP implied the increase of the half time for wound recovery migration. Together, our results demonstrate that partial uncoupling of mitochondrial oxidative phosphorylation reduces micromotion and wound healing migration of hMSCs. The ECIS method used in this study offers a simple and sensitive approach to investigate stem cell migration and its regulation by mitochondrial dynamics.

A reliable transmission protocol for ZigBee-based wireless patient monitoring.

Patient monitoring systems are gaining their importance as the fast-growing global elderly population increases demands for caretaking. These systems use wireless technologies to transmit vital signs for medical evaluation. In a multihop ZigBee network, the existing systems usually use broadcast or multicast schemes to increase the reliability of signals transmission; however, both the schemes lead to significantly higher network traffic and end-to-end transmission delay. In this paper, we present a reliable transmission protocol based on anycast routing for wireless patient monitoring. Our scheme automatically selects the closest data receiver in an anycast group as a destination to reduce the transmission latency as well as the control overhead. The new protocol also shortens the latency of path recovery by initiating route recovery from the intermediate routers of the original path. On the basis of a reliable transmission scheme, we implement a ZigBee device for fall monitoring, which integrates fall detection, indoor positioning, and ECG monitoring. When the triaxial accelerometer of the device detects a fall, the current position of the patient is transmitted to an emergency center through a ZigBee network. In order to clarify the situation of the fallen patient, 4-s ECG signals are also transmitted. Our transmission scheme ensures the successful transmission of these critical messages. The experimental results show that our scheme is fast and reliable. We also demonstrate that our devices can seamlessly integrate with the next generation technology of wireless wide area network, worldwide interoperability for microwave access, to achieve real-time patient monitoring.