High-throughput single-cell quantification using simple microwell-based cell docking and programmable time-course live-cell imaging

Extracting single-cell information during cellular responses to external signals in a high-throughput manner is an essential step for quantitative single-cell analyses. Here, we have developed a simple yet robust microfluidic platform for measuring time-course single-cell response on a large scale. Our method combines a simple microwell-based cell docking process inside a patterned microfluidic channel, with programmable time-course live-cell imaging and software-aided fluorescent image processing. The budding yeast, Saccharomyces cerevisiae (S. cerevisiae), cells were individually captured in microwells by multiple sweeping processes, in which a cell-containing solution plug was actively migrating back and forth several times by a finger-pressure induced receding meniscus. To optimize cell docking efficiency while minimizing unnecessary flooding in subsequent steps, circular microwells of various channel dimensions (4-24 μm diameter, 8 μm depth) along with different densities of cell solution (1.5-6.0 × 10(9) cells per mL) were tested. It was found that the microwells of 8 μm diameter and 8 μm depth allowed for an optimal docking efficiency (>90%) without notable flooding issues. For quantitative single-cell analysis, time-course (time interval 15 minute, for 2 hours) fluorescent images of the cells stimulated by mating pheromone were captured using computerized fluorescence microscope and the captured images were processed using a commercially available image processing software. Here, real-time cellular responses of the mating MAPK pathway were monitored at various concentrations (1 nM-100 μM) of mating pheromone at single-cell resolution, revealing that individual cells in the population showed non-uniform signaling response kinetics.     

Comparison of Cytotoxic Changes in ECV304 Cell Through Exposure to DMSO and Sodium Butyrate Using Impedance Spectroscopy

This paper decides an impedance spectroscopy measurement system for real-time monitoring of cellular toxicity. The system is used to compare the impedance response of ECV304 cells to two toxicants, dimethyl sulphoxide (DMSO) and sodium butyrate. The responses at different concentration of toxicants were plotted in 3-D impedance spectra and their correlation coefficients were calculated at four time points. Additionally, Phase contrast microscopy images were used to observe the effect of the two toxicants on the morphology change of the cells. The characteristic information of the spectra compared well with the morphological changes of cells, illustrating that the method is useful for monitoring cytotoxicity effects and cell viability.     

Single-cell recording and stimulation with a 16k micro-nail electrode array integrated on a 0.18 μm CMOS chip

To cope with the growing needs in research towards the understanding of cellular function and network dynamics, advanced micro-electrode arrays (MEAs) based on integrated complementary metal oxide semiconductor (CMOS) circuits have been increasingly reported. Although such arrays contain a large number of sensors for recording and/or stimulation, the size of the electrodes on these chips are often larger than a typical mammalian cell. Therefore, true single-cell recording and stimulation remains challenging. Single-cell resolution can be obtained by decreasing the size of the electrodes, which inherently increases the characteristic impedance and noise. Here, we present an array of 16,384 active sensors monolithically integrated on chip, realized in 0.18 μm CMOS technology for recording and stimulation of individual cells. Successful recording of electrical activity of cardiac cells with the chip, validated with intracellular whole-cell patch clamp recordings are presented, illustrating single-cell readout capability. Further, by applying a single-electrode stimulation protocol, we could pace individual cardiac cells, demonstrating single-cell addressability. This novel electrode array could help pave the way towards solving complex interactions of mammalian cellular networks.     

Potentiometric platform for the quantification of cellular potassium efflux

Renewed interest in the measurement of cellular K(+) effluxes has been prompted by the observation that potassium plays an active and important role in numerous key cellular events, in particular cell necrosis and apoptosis. Although necrosis and apoptosis follow different pathways, both induce intracellular potassium effluxes. Here, we report the use of potassium-selective microelectrodes located in a microfluidic platform for cell culture to monitor and quantify such effluxes in real time. Using this platform, we observed and measured the early signs of cell lysis induced by a modification of the extracellular osmolarity. Furthermore, we were able to quantify the number of dying cells by evaluating the extracellular potassium concentration. A comparison between the potentiometric measurement with a fluorescent live-dead assay performed under similar conditions revealed the delay between potassium effluxes and cell necrosis. These results suggest that such platforms may be exploited for applications, such as cytotoxicological screening assays or tumor cell proliferation assays, by using extracellular K(+) as cell death marker.     

Effects of electrode surface modification with chlorotoxin on patterning single glioma cells

A microchip patterned with arrays of single cancer cells can be an effective platform for the study of tumor biology, medical diagnostics, and drug screening. However, patterning and retaining viable single cancer cells on defined sites of the microarray can be challenging. In this study we used a tumor cell-specific peptide, chlorotoxin (CTX), to mediate glioma cell adhesion on arrays of gold microelectrodes and investigated the effects of three surface modification schemes for conjugation of CTX to the microelectrodes on single cell patterning, which include physical adsorption, covalent bonding mediated by N-hydroxysuccinimide (NHS), and covalent bonding via crosslinking succinimidyl iodoacetate and Traut's (SIA-Traut) reagents. The CTX immobilization to microelectrodes was confirmed by high-resolution X-ray photoelectron spectroscopy. Physically adsorbed CTX showed better support for cell adhesion and is more effective in confining adhered cells on the electrodes than covalently-bound CTX. Furthermore, cell adhesion and spreading on microelectrodes were quantified in real-time by impedance measurements, which revealed an impedance signal from physically adsorbed CTX electrodes four times greater than the signal from covalently-bound CTX electrodes.     

Hydrogen Peroxide Detection Using Prussian Blue-modified 3D Pyrolytic Carbon Microelectrodes

A highly sensitive amperometric Prussian blue-based hydrogen peroxide sensor was developed using 3D pyrolytic carbon microelectrodes. A 3D printed multielectrode electrochemical cell enabled simultaneous highly reproducible Prussian blue modification on multiple carbon electrodes. The effect of oxygen plasma pre-treatment and deposition time on Prussian blue electrodeposition was studied. The amperometric response of 2D and 3D sensors to the addition of hydrogen peroxide in μM and sub-μM concentrations in phosphate buffer was investigated. A high sensitivity comparable to flow injection systems and a detection limit of 0.16 μM was demonstrated with 3D pyrolytic carbon microelectrodes at stirred batch condition     

Oxidation-dependent effects of oxidized LDL: proliferation or cell death

Oxidized low-density lipoprotein (oxLDL) induces a wide range of cellular responses to produce atherosclerotic lesion, but key factors determining the response are not understood. In this study, purified LDL was oxidized with copper sulfate, and its physical properties and the related biological responses were investigated. The average hydrodynamic diameter of the lightly oxidized LDL was approximately 25 nm and its Rf value relative to nLDL on agarose gel was between 1.0 and 1.25. The diameter of the extensively oxidized LDL was over 30 nm, the Rf value was over 2.0. A 24 h-exposure of resting RAW264.7 macrophage cells to 100 microg/ml of the lightly oxidized LDL induced proliferation and macrophage activation whereas the extensively oxidized LDL induced cell death at the same concentration. In contrast, 200 microg/ml of oxLDL caused cell death regardless of oxidation degree. Short incubation (4-6 h) of the highly oxidized LDL (100 microg/ml) also resulted in cell proliferation. OxLDL-induced cell death showed mixed characteristics of apoptosis and/or necrosis depending on the strength and duration of the insult. These results suggest that cellular responses induced by oxLDL be dependent on the oxidation degree, the duration of exposure, and the concentration of oxLDL.     

From Oncogenic Signaling Pathways to Single-Cell Sequencing of Immune Cells: Changing the Landscape of Cancer Immunotherapy

Over the past decade, there have been remarkable advances in understanding the signaling pathways involved in cancer development. It is well-established that cancer is caused by the dysregulation of cellular pathways involved in proliferation, cell cycle, apoptosis, cell metabolism, migration, cell polarity, and differentiation. Besides, growing evidence indicates that extracellular matrix signaling, cell surface proteoglycans, and angiogenesis can contribute to cancer development. Given the genetic instability and vast intra-tumoral heterogeneity revealed by the single-cell sequencing of tumoral cells, the current approaches cannot eliminate the mutating cancer cells. Besides, the polyclonal expansion of tumor-infiltrated lymphocytes in response to tumoral neoantigens cannot elicit anti-tumoral immune responses due to the immunosuppressive tumor microenvironment. Nevertheless, the data from the single-cell sequencing of immune cells can provide valuable insights regarding the expression of inhibitory immune checkpoints/related signaling factors in immune cells, which can be used to select immune checkpoint inhibitors and adjust their dosage. Indeed, the integration of the data obtained from the single-cell sequencing of immune cells with immune checkpoint inhibitors can increase the response rate of immune checkpoint inhibitors, decrease the immune-related adverse events, and facilitate tumoral cell elimination. This study aims to review key pathways involved in tumor development and shed light on single-cell sequencing. It also intends to address the shortcomings of immune checkpoint inhibitors, i.e., their varied response rates among cancer patients and increased risk of autoimmunity development, via applying the data from the single-cell sequencing of immune cells.     

Microelectrodes for the measurement of cellular metabolism

The study of metabolism of cellular cultures is of great interest. Although some papers reports the monitoring of parameters such as pH, potassium, dissolved oxygen and temperature in cellular cultures using different kind of sensors, no definitive solution has been found to get reproducible and feasible results. The advantage of using sensors fabricated with microelectronic technology is focused on their small size, rapid response and the possibility of having an integrated array of microelectrodes, which favors the measurement of small volumes and fast cell metabolism changes. In this work, the viability of using a multisensor platform of microelectrodes, such as potassium and pH ISFETs, amperometric microelectrodes for dissolved O₂ and pseudo-reference microelectrodes for the study of metabolism of cellular cultures is described. Preliminary results of pH ISFETS’ response in neuronal cultures is reported.     

Multiplexing microelectrodes for dielectrophoretic manipulation and electrical impedance measurement of single particles and cells in a microfluidic device

This work presents a microfluidic device, which was patterned with (i) microstructures for hydrodynamic capture of single particles and cells, and (ii) multiplexing microelectrodes for selective release via negative dielectrophoretic (nDEP) forces and electrical impedance measurements of immobilized samples. Computational fluid dynamics (CFD) simulations were performed to investigate the fluidic profiles within the microchannels during the hydrodynamic capture of particles and evaluate the performance of single‐cell immobilization. Results showed uniform distributions of velocities and pressure differences across all eight trapping sites. The hydrodynamic net force and the nDEP force acting on a 6 μm sphere were calculated in a 3D model. Polystyrene beads with difference diameters (6, 8, and 10 μm) and budding yeast cells were employed to verify multiple functions of the microfluidic device, including reliable capture and selective nDEP‐release of particles or cells and sensitive electrical impedance measurements of immobilized samples. The size of immobilized beads and the number of captured yeast cells can be discriminated by analyzing impedance signals at 1 MHz. Results also demonstrated that yeast cells can be immobilized at single‐cell resolution by combining the hydrodynamic capture with impedance measurements and nDEP‐release of unwanted samples. Therefore, the microfluidic device integrated with multiplexing microelectrodes potentially offers a versatile, reliable, and precise platform for single‐cell analysis.     

Dynamic metabolic change of cancer cells induced by natural killer cells at the single-cell level studied by label-free mass cytometry

Natural killer cells (NK cells) are important immune cells which have attracted increasing attention in cancer immunotherapy. Due to the heterogeneity of cells, individual cancer cells show different resistance to NK cytotoxicity, which has been revealed by flow cytometry. Here we used label-free mass cytometry (CyESI-MS) as a new tool to analyze the metabolites in Human Hepatocellular Carcinoma (HepG2) cells at the single-cell level after the interaction with different numbers of NK92 MI cells. A large amount of chemical information from individual HepG2 cells was obtained showing the process of cell apoptosis induced by NK cells. Nineteen metabolites which consecutively change during cell apoptosis were revealed by calculating their average relative intensity. Four metabolic pathways were impacted during cell apoptosis which hit 4 metabolites including glutathione (GSH), creatine, glutamic acid and taurine. We found that the HepG2 cells could be divided into two phenotypes after co-culturing with NK cells according to the bimodal distribution of concentration of these 4 metabolites. The correlation between metabolites and different apoptotic pathways in the early apoptosis cell group was established by the 4 metabolites at the single-cell level. This is a new idea of using single-cell specific metabolites to reveal the metabolic heterogeneity in cell apoptosis which would be a powerful means for evaluating the cytotoxicity of NK cells.

Label-free mass cytometry is utilized to study the dynamic metabolic change during apoptosis in HepG2 cells induced by NK92 MI cells at the single-cell level. The metabolic heterogeneity of individual HepG2 cells during apoptosis was revealed.     

Electrical impedance spectroscopy for detection of bacterial cells in suspensions using interdigitated microelectrodes

In this study, we present a new, simple and rapid impedance method to detect bacterial cells by making use of the impedance properties of bacterial cell suspensions using interdigitated microelectrodes. It was found that bacterial cell suspensions in deionized (DI) water with different cell concentrations could generate different electrical impedance spectral responses, whereas cell suspensions in phosphate buffered saline (PBS) solution could not produce any significant differences in impedance spectra in response to different cell concentrations. In DI water suspensions, impedance at 1 kHz decreased with the increasing cell concentrations in the suspensions. The impedance of cell suspensions in DI water was discussed and found that it was resulted from the cell wall charges and the release of ions or other osmolytes from the cells. A linear relationship between the impedance and the logarithmic value of the cell concentration was found in the cell concentration range from 10⁶ to 10¹⁰ cfu/ml, which can be expressed by a regression equation of Z (kΩ) = −2.06 log C (cells/20 μl) + 5.23 with R² = 0.98. The detection limit was calculated to be 3.45 × 10⁶ cfu/ml, which is comparable with many label-free immunosensors for detection of pathogenic bacteria reported in the literature. To achieve the selectivity of this method, we also demonstrated the feasibility of integrating magnetic separation to this impedance method. This study has demonstrated that bacterial cell concentration can be inferred by measuring the impedance of cell suspensions in DI water. This new detection mechanism could be an alternative to current impedance methods that have been reported for the detection of bacterial cells, e.g. impedance microbiology and electrical/electrochemical impedance biosensors.     

Purpurin-18 in combination with light leads to apoptosis or necrosis in HL60 leukemia cells

Photodynamic therapy (PDT), a cancer treatment using a photosensitizer and visible light, has been shown to induce apoptosis or necrosis. We report here that Purpurin-18 (Pu18) in combination with light induces rapid apoptotic cell death in the human leukemia cell line (HL60) at low doses and necrosis at higher concentrations. Cells treated with Pu18 and light under apoptotic conditions exhibited DNA laddering and an increase in both cellular content of subdiploid DNA and externalization of phosphatidylserine (PS), indicating DNA fragmentation and loss of membrane phospholipid asymmetry. In the absence of light activation, Pu18 at nanomolar concentrations had no detectable cytotoxic effect. Caspase-3 activity was increased even after 1 h from treatment with low doses of Pu18 and light. The PS exposure and nuclear features of apoptosis were prevented by treatment of cells before illumination with caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK). Conversely, the caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO) failed to suppress the apoptosis. No protective effect of the three caspase inhibitors was observed when the cells were exposed to necrotic concentrations of Pu18 and light. Our results show that caspase-3, but not caspase-1, is involved in the signaling of apoptotic events in PDT with Pu18-induced apoptosis of HL60 cells. Moreover, both the time course of PS exposure and the effect of caspase inhibitors on it indicate that it is regulated in the same manner as DNA fragmentation.     

Short peptides enhance single cell adhesion and viability on microarrays

Single cell patterning holds important implications for biology, biochemistry, biotechnology, medicine, and bioinformatics. The challenge for single cell patterning is to produce small islands hosting only single cells and retaining their viability for a prolonged period of time. This study demonstrated a surface engineering approach that uses a covalently bound short peptide as a mediator to pattern cells with improved single cell adhesion and prolonged cellular viability on gold patterned SiO2 substrates. The underlying hypothesis is that cell adhesion is regulated by the type, availability, and stability of effective cell adhesion peptides, and thus covalently bound short peptides would promote cell spreading and, thus, single cell adhesion and viability. The effectiveness of this approach and the underlying mechanism for the increased probability of single cell adhesion and prolonged cell viability by short peptides were studied by comparing cellular behavior of human umbilical cord vein endothelial cells on three model surfaces whose gold electrodes were immobilized with fibronectin, physically adsorbed Arg-Glu-Asp-Val-Tyr, and covalently bound Lys-Arg-Glu-Asp-Val-Tyr, respectively. The surface chemistry and binding properties were characterized by reflectance Fourier transform infrared spectroscopy. Both short peptides were superior to fibronectin in producing adhesion of only single cells, whereas the covalently bound peptide also reduced apoptosis and necrosis of adhered cells. Controlling cell spreading by peptide binding domains to regulate apoptosis and viability represents a fundamental mechanism in cell-materials interaction and provides an effective strategy in engineering arrays of single cells.     

Signaling pathways in cell death and survival after photodynamic therapy

Photodynamic therapy (PDT) is a cytotoxic treatment, which can induce cells to initiate a rescue response, or to undergo cell death, either apoptosis or necrosis. The many signaling pathways involved in these processes are the topic of this review. The subcellular localization of the photosensitizer has been shown to be a key factor in the outcome of PDT. Mitochondrial localized photosensitizers are able to induce apoptosis very rapidly. Lysosomal localized photosensitizers can elicit either a necrotic or an apoptotic response. In the plasma membrane, a target for various photosensitizers, rescue responses, apoptosis and necrosis is initiated. Several protein phosphorylation cascades are involved in the regulation of the response to PDT. Finally, a number of stress-induced proteins play a role in the rescue response after PDT. Notably, the induction of apoptosis by PDT might not be crucial for an optimal outcome. Recent studies indicate that abrogation of the apoptotic pathway does alter the clonogenic survival of the cells after PDT. Further studies, both in vitro and especially in vivo could lead to more efficient combination therapies in which signaling pathways, involved in cell death or rescue, are either up- or downregulated before PDT.     

Micromachined impedance spectroscopy flow cytometer for cell analysis and particle sizing

A new cytological tool, based on the microCoulter particle counter (microCPC) principle, aimed at diagnostic applications for cell counting and separation in haematology, oncology or toxicology is described. The device measures the spectral impedance of individual cells or particles and allows screening rates over 100 samples s(-1) on a single-cell basis. This analyzer is intended to drive a sorting actuator producing a subsequent cell separation. Size reduction and integration of functions are essential in achieving precise measurements and high throughput. 3D finite element simulations are presented to compare various electrode geometries and their influence on cell parameters estimation. The device is based on a glass-polyimide microfluidic chip with integrated channels and electrodes microfabricated at the length scale of the particles to be investigated (1-20 microm). A laminar liquid flow carries the suspended particles through the measurement area. Each particle's impedance signal is recorded by a differential pair of microelectrodes using the cell surrounding media as a reference. The micromachined chip and processing electronic circuit allow simultaneous impedance measurements at multiple frequencies, ranging from 100 kHz to 15 MHz. In this paper, we describe the microfabrication and characterisation of an on-chip flow-cytometer as the first building block of a complete cell-sorting device. We then discuss the signal conditioning technique and finally impedance measurements of cells and particles of different sizes and types to demonstrate the differentiation of subpopulations in a mixed sample.     

Single-cell metabolite analysis on a microfluidic chip

Metabolites can directly reflect and modulate cell responses and phenotypical changes by influencing energy balances, intercellular signals, and many other cellular functions throughout the lifespan of cells.Taking into account the heterogeneity of cells, single-cell metabolite analysis offers an insight into the functional process within one cell. Microfluidics as a powerful tool has attracted significant interest in the single-cell metabolite analysis field. The microfluidic platform is possib...     

Apoptotic cell death dynamics of HL60 cells studied using a microfluidic cell trap device

This paper presents the design, fabrication and first results of a microfluidic cell trap device for analysis of apoptosis. The microfluidic silicon-glass chip enables the immobilization of cells and real-time monitoring of the apoptotic process. Induction of apoptosis, either electric field mediated or chemically induced with tumour necrosis factor (TNF-alpha), in combination with cycloheximide (CHX), was addressed. Exposure of cells to the appropriate fluorescent dyes, FLICA and PI, allows one to discriminate between viable, apoptotic and necrotic cells. The results showed that the onset of apoptosis and the transitions during the course of the cell death cascade were followed in chemically induced apoptotic HL60 cells. For the case of electric field mediated cell death, the distinction between apoptotic and necrotic stage was not clear. This paper presents the first results to analyse programmed cell death dynamics using this apoptosis chip and a first step towards an integrated apoptosis chip for high-throughput drug screening on a single cellular level.     

Cellular impedance biosensors for drug screening and toxin detection

Cell-based impedance biosensing is an emerging technology that can be used to non-invasively and instantaneously detect and analyze cell responses to chemical and biological agents. This article highlights the fabrication and measurement technologies of cell impedance sensors, and their application in toxin detection and anti-cancer drug screening. We start with an introduction that describes the capability and advantages of cell-based sensors over conventional sensing technology, followed by a discussion of the influence of cell adhesion, spreading and viability during cell patterning on the subsequent impedance measurements and sensing applications. We then present an electronic circuit that models the cell-electrode system, by which the cellular changes can be detected in terms of impedance changes of the circuit. Finally, we discuss the current status on using cell impedance sensors for toxin detection and anti-cancer drug screening.     

Real-time cell-microelectronic sensing of nanoparticle-induced cytotoxic effects

We report a real-time cell analysis (RTCA) sensing method of 96 electronic microwells for profiling the cytotoxicity of nanoparticles on different cell lines. The method consists of 96 microwells embedded with microelectrodes (96x E-plate) to measure impedance changes of adherent cell lines. When the testing cells change in population, adhesion, and/or morphology, the impedance at the cell–electrode interface changes to provide real-time monitoring of overall cell status. To demonstrate this technique, we used three cell lines as sensing probes: two human lung carcinoma cell lines, A549 and SK-MES-1, and a normal mammalian cell line, CHO-K1. We tested two well-characterized nanoparticles: nano-titanium dioxide (nTiO₂) and nano-silver (nAg). The three cell lines were separately seeded into 96x E-plates and treated with varying concentrations of nanoparticles (0.078–160 μg mL⁻¹). This method provides dynamic cell response profiles and temporal IC₅₀ histograms, showing concentration-, time-, particle-, and cell-dependent cytotoxicity. The 24 h and 48 h IC₅₀ values of nAg obtained using both the RTCA and the neutral red uptake (NRU) assays were in good agreement, validating the RTCA technique. The RTCA assay does not suffer interference from nTiO₂, whereas the NRU assay cannot be used due to severe interference from nTiO₂. A cytostatic response was observed in CHO-K1 cells after 24 h exposure to 40 μg mL⁻¹ nTiO₂, which was correlated with S-phase cell cycle arrest based on cell cycle analysis using flow cytometry. This suggests that the shapes of the response curves provide indicative information, directing further studies into the mode of action of the toxicant. Advantages of the RTCA technique over traditional colorimetric assays for screening the cytotoxicity of nanoparticles include minimizing interference, qualitative and quantitative cytotoxicity data, and the capability of real-time and high-throughput measurements.