A droplet-merging platform for comparative functional analysis of m1 and m2 macrophages in response to e. coli-induced stimuli.

Microfluidic droplets are used to isolate cell pairs and prevent crosstalk with neighboring cells, while permitting free motility and interaction within the confined space. Dynamic analysis of cellular heterogeneity in droplets has provided insights in various biological processes. Droplet manipulation methods such as fusion and fission make it possible to precisely regulate the localized environment of a cell in a droplet and deliver reagents as required. Droplet fusion strategies achieved by passive mechanisms preserve cell viability and are easier to fabricate and operate. Here, we present a simple and effective method for the co-encapsulation of polarized M1 and M2 macrophages with Escherichia coli (E. coli) by passive merging in an integrated droplet generation, merging, and docking platform. This approach facilitated live cell profiling of effector immune functions in situ and quantitative functional analysis of macrophage heterogeneity. Biotechnol. Bioeng. 2017;114: 705-709. © 2016 Wiley Periodicals, Inc.

Biosensing approaches for rapid genotoxicity and cytotoxicity assays upon nanomaterial exposure.

The increased utilization of nanomaterials could affect human health and the environment due to increased exposure. Several mechanisms regarding the negative effects of nanomaterials have been proposed, one of the most discussed being oxidative stress. Many studies have shown that some metal oxide nanoparticles can enhance reactive oxygen species generation, inducing oxidative stress, DNA damage, and unregulated cell signaling, and eventually leading to changes in cell motility, apoptosis, and even carcinogenesis. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is one of the predominant forms of oxidative DNA damage, and has therefore been widely used as a biomarker for oxidative stress and carcinogenesis. Ther are two major objectives to this study. Firstly, the development of a novel lateral flow immunoassay (LFIA) is presented to measure the concentration of 8-OHdG in cells and thus reveal the nanotoxicity on the genomic level. The feasibility of this new method is validated by comparison with two other established methods: Alamar Blue assay and a recently developed electrical impedance sensing (EIS) system on the level of cell proliferation/viability. Secondly, the toxicological effects of three metallic nanoparticles (CuO, CdO, and TiO2 ) are investigated and compared using these three methods with completely different mechanisms. The results show that there is a high variation among different nanoparticles concerning their ability to cause toxic effects. CuO nanoparticles are the most potent regarding cytotoxicity and DNA damage. CdO shows a fallen cell viability as well as DNA damage, however, to a lesser extent than CuO nanoparticles. TiO2 particles only cause very limited cytotoxicity, and there is no obvious increase in 8-OHdG levels. In conclusion, LFIA as well as the EIS system are useful methods for quantitative or qualitative nanotoxicity assessments with high sensitivity, specificity, speed of performance, and simplicity.

Electrical field manipulation of cancer cell behavior monitored by whole cell biosensing device.

All living cells possess electrical characteristics and are thus responsive to, and even generate electric fields and currents. It has been shown that the electrical properties of cancer cells differ from normal proliferating cells, thus electric fields may induce differential effects in normal and cancer cells. Manipulation of these electrical properties may provide a powerful direct and/or adjuvant therapeutic option for cancer. A whole cell impedance-based biosensor to monitor the effects of a range of different frequencies (50 kHz-2 MHz) at low-intensity (<2 V/cm) on the growth rate of human SKOV3 ovarian cancer cells versus non-cancerous HUVECs is reported. Rapid real-time monitoring of the SKOV3 behavior was observed as the alternating electric fields were applied and the impedimetric response of the cells was recorded. The cells were also labeled with propidium iodide to examine morphological changes and cell viability with fluorescence microscopy with trypan blue for comparison. A noticeable decrease in the growth profile of the SKOV3 was observed with the application of 200 kHz alternating electric fields indicating specific inhibitory effects on dividing cells in culture in contrast to the HUVECs. The outcome of this research will improve our fundamental understanding of the behavior of cancer cells when exposed to alternating electric fields at specific frequencies and foster the development strategies and optimal parameters for alternating electric field therapies for clinical and drug delivery applications.

Whole cell based electrical impedance sensing approach for a rapid nanotoxicity assay.

A whole cell based biosensor for rapid real-time testing of human and environmental toxicity of nanoscale materials is reported. Recent studies measuring nanoparticle cytotoxicity in vitro provide a final measurement of toxicity to a cell culture overlooking the ongoing cytotoxic effects of the nanoparticles over the desired timeframe. An array biosensor capable of performing multiple cytotoxicity assays simultaneously was designed to address the need for a consistent method to measure real-time assessments of toxicity. The impedimetric response of human lung fibroblasts (CCL-153) and rainbow trout gill epithelial cells (RTgill-W1) when exposed to gold and silver nanoparticles (AuNPs, AgNPs), single walled carbon nanotubes (SWCNTs) and cadmium oxide (CdO) was tested. Exposure to CdO particles exhibited the fastest rate of cytotoxicity and demonstrated the biosensor's ability to monitor toxicity instantaneously in real time. Advantages of the present method include shorter run times, easier usage, and multi-sample analysis leading to a method that can monitor the kinetic effects of nanoparticle toxicity continuously over a desired timeframe.

Quantification of intercellular adhesion forces measured by fluid force microscopy.

The mechanics of cancer cell adhesion to its neighboring cells, homotypic or heterotypic, have significant impact on tumor progression and metastasis. Intercellular adhesion has been quantified previously using atomic force microscopy-based methods. Here we show the feasibility of the recently developed fluidic force microscopy (FluidFM) to measure adhesive forces exerted by breast cancer cells. Multiple cell pairs were assessed at precisely controlled, increasing contact durations by pressure-dependent immobilization of a cell at the probe tip. Eliminating chemical fixation of the cell at the tip ensured repeated use of the same probe and also minimized changes in cell physiology. Our data indicates distinct trends of adhesion forces between homotypic breast cancer cells compared to heterotypic adhesion between cancer-fibroblast and cancer-epithelial cell pairs. Adhesion forces were similar for all three cell pairs at short contact duration (< 1min) but differed at longer contact period (30min). Our study suggests that FluidFM is a rapid efficient technique that could be used to assess heterogeneity in cellular adhesion at various stages of malignant transformation.

Immuno nanoparticles integrated electrical control of targeted cancer cell development using whole cell bioelectronic device.

Electrical properties of cells determine most of the cellular functions, particularly ones which occur in the cell's membrane. Manipulation of these electrical properties may provide a powerful electrotherapy option for the treatment of cancer as cancerous cells have been shown to be more electronegative than normal proliferating cells. Previously, we used an electrical impedance sensing system (EIS) to explore the responses of cancerous SKOV3 cells and normal HUVEC cells to low intensity (<2 V/cm) AC electric fields, determining that the optimal frequency for SKOV3 proliferation arrest was 200 kHz, without harming the non-cancerous HUVECs. In this study, to determine if these effects are cell type dependant, human breast adenocarcinoma cells (MCF7) were subjected to a range of frequencies (50 kHz-2 MHz) similar to the previously tested SKOV3. For the MCF7, an optimal frequency of 100 kHz was determined using the EIS, indicating a higher sensitivity towards the applied field. Further experiments specifically targeting the two types of cancer cells using HER2 antibody functionalized gold nanoparticles (HER2-AuNPs) were performed to determine if enhanced electric field strength can be induced via the application of nanoparticles, consequently leading to the killing of the cancerous cells without affecting non cancerous HUVECs and MCF10a providing a platform for the development of a non-invasive cancer treatment without any harmful side effects. The EIS was used to monitor the real-time consequences on cellular viability and a noticeable decrease in the growth profile of the MCF7 was observed with the application of the HER2-AuNPs and the electric fields indicating specific inhibitory effects on dividing cells in culture. To further understand the effects of the externally applied field to the cells, an Annexin V/EthD-III assay was performed to determine the cell death mechanism indicating apoptosis. The zeta potential of the SKOV3 and the MCF7 before and after incorporation of the HER2-AuNPs was also obtained indicating a decrease in zeta potential with the incorporation of the nanoparticles. The outcome of this research will improve our fundamental understanding of the behavior of cancer cells and define optimal parameters of electrotherapy for clinical and drug delivery applications.

Whole cell impedance biosensoring devices.

Nanotechnology is rapidly growing and has great potential in various fields such as biomedical engineering, drug delivery, environmental health, pharmaceutical industries and even electronics and communication technologies. However, with this rapid development, these new nanoscale materials (including nanotubes, nanowires, nanowhiskers, fullerenes or buckyballs, and quantum dots) might have unintended human health and environmental hazards. Testing for toxicological parameters is a necessary first step toward ensuring the compatibility of nanomaterials for medical applications and for the safety of the environment. Here, we describe an array formatted electrical impedance sensing (EIS) system that is capable of measuring nanotoxicity in real time.