Dr Fatima Labeed

Circadian rhythms are biological adaptations to the day-night cycle whereby cells adapt to changes in the external environment or internal physiology according to the time of day. Whilst many cellular clock mechanisms involve gene expression feedback mechanisms, clocks operate even where gene expression is absent. For example, red blood cells (RBCs) do not have capacity for gene expression, and instead, possess an electrophysiological oscillator where cytosolic potassium plays a key role in timekeeping. Here, we examined murine blood under normal conditions as well as in two perturbed states, malaria infection and induced anemia, to assess changes in baseline cellular electrophysiology and its implications for the electrophysiological oscillator. The ex-vivo blood samples were analyzed at four-hour intervals over two days by dielectrophoresis, and microscopic determination of parasitemia. We found that cytoplasmic conductivity (indicating the concentration of free ions in the cytoplasm and related to the membrane potential) exhibited circadian rhythmic behavior in all three cases (control, malaria and anemia). Compared to control samples, cytoplasm conductivity was decreased in the anemia group, whilst malaria-infected samples were in antiphase to control. Furthermore, we identified rhythmic behavior in membrane capacitance of malarial cells that was not replicated in the other samples. Finally, we reveal the historically famous rhythmicity of malaria parasite replication is in phase with cytoplasm conductivity. Our findings suggest the electrophysiological oscillator can impact on malaria parasite replication and/or is vulnerable to perturbation by rhythmic parasite activities.

Even in nonexcitable cells, the membrane potential Vm is fundamental to cell function, with roles from ion channel regulation, development, to cancer metastasis. Vm arises from transmembrane ion concentration gradients; standard models assume homogeneous extracellular and intracellular ion concentrations, and that Vm only exists across the cell membrane and has no significance beyond it. Using red blood cells, we show that this is incorrect, or at least incomplete; Vm is detectable in the extracellular ion concentration beyond the cell surface, and that modulating Vm produces quantifiable and consistent changes in extracellular potential. Evidence strongly suggests this is due to capacitive coupling between Vm and the electrical double layer, rather than molecular transporters. We show that modulating Vm changing the extracellular ion composition mimics the behaviour of voltage-activated ion channel in non-excitable channels. We also observe Vm-synchronised circadian rhythms in extracellular potential, with significant implications for cell-cell interactions and cardiovascular disease.

Delays in the identification and referral of oral cancer remain frequent. An accurate and non-invasive diagnostic test to be performed in primary care may help identifying oral cancer at an early stage and reduce mortality. PANDORA was a proof-of-concept prospective diagnostic accuracy study aimed at advancing the development of a dielectrophoresis-based diagnostic platform for oral squamous cell carcinoma (OSCC) and epithelial dysplasia (OED) using a novel automated DEPtech 3DEP analyser. the aim of PANDORA was to identify the set-up of the DEPtech 3DEP analyser associated with the highest diagnostic accuracy in identifying OSCC and OED from non-invasive brush biopsy samples, as compared to the gold standard test (histopathology). Measures of accuracy included sensitivity, specificity, positive and negative predictive value. Brush biopsies were collected from individuals with histologically-proven OSCC and OED, histologically-proven benign mucosal disease, and healthy mucosa (standard test), and analysed via dielectrophoresis (index test). 40 individuals with OSCC/OED and 79 with benign oral mucosal disease/healthy mucosa were recruited. Sensitivity and specificity of the index test was 86.8% (95% CI, 71.9%-95.6%) and 83.6% (95% CI, 73.0%-91.2%). Analysing OSCC samples separately led to higher diagnostic accuracy, with 92.0% (95% CI, 74.0%-99.0%) sensitivity and 94.5% (95% CI, 86.6%-98.5%) specificity. The DEPtech 3DEP analyser has the potential to identify OSCC and OED with notable diagnostic accuracy and warrants further investigation as a potential triage test in the primary care setting for patients who may need to progress along the diagnostic pathway and be offered a surgical biopsy. This article is protected by copyright. All rights reserved.

Dielectrophoresis (DEP) enables the measurement of population-level electrophysiology in many cell types by examining their interaction with an externally applied electric field. Here we describe the application of DEP to the measurement of circadian rhythms in a non-nucleated cell type, the human red blood cell. Using DEP, population-level electrophysiology of ~20,000 red blood cells can be measured from start to finish in less than 3 min, and can be repeated over several days to reveal cell-autonomous daily regulation of membrane electrophysiology. This method is amenable to the characterization of circadian rhythms by altering entrainment and free-run conditions or through pharmacological perturbation.

A loss of ability of cells to undergo apoptosis (programmed cell death, whereby the cell ceases to function and destroys itself) is commonly associated with cancer, and many anti-cancer interventions aim to restart the process. Consequently, the accurate quantification of apoptosis is essential in understanding the function and performance of new anti-cancer drugs. Dielectrophoresis has previously been demonstrated to detect apoptosis more rapidly than other methods, and is low-cost, label-free and rapid, but has previously been unable to accurately quantify cells through the apoptotic process because cells in late apoptosis disintegrate, making cell tracking impossible. In this paper we use a novel method based on light absorbance and multi-population tracking to quantify the progress of apoptosis, benchmarking against conventional assays including MTT, trypan blue and Annexin-V. Analyses are performed on suspension and adherent cells, and using two apoptosis-inducing agents. IC50 measurements compared favourably to MTT and were superior to trypan blue, whilst also detecting apoptotic progression faster than Annexin-V.

© 2012 John Wiley & Sons, Ltd. Dielectrophoresis (DEP) is a non-invasive cell analysis method that uses differences in electrical properties between particles and surrounding medium to determine a unique set of cellular properties that can be used as a basis for cell separation. Cell-based therapies using skeletal stem cells are currently one of the most promising areas for treating a variety of skeletal and muscular disorders. However, identifying and sorting these cells remains a challenge in the absence of unique skeletal stem cell markers. DEP provides an ideal method for identifying subsets of cells without the need for markers by using their dielectric properties. This study used a 3D dielectrophoretic well chip device to determine the dielectric characteristics of two osteosarcoma cell lines (MG-63 and SAOS-2) and an immunoselected enriched skeletal stem cell fraction (STRO-1 positive cell) of human bone marrow. Skeletal cells were exposed to a series of different frequencies to induce dielectrophoretic cell movement, and a model was developed to generate the membrane and cytoplasmic properties of the cell populations. Differences were observed in the dielectric properties of MG-63, SAOS-2 and STRO-1 enriched skeletal populations, which could potentially be used to sort cells in mixed populations. This study provide evidence of the ability to characterize different human skeletal stem and mature cell populations, and acts as a proof-of-concept that dielectrophoresis can be exploited to detect, isolate and separate skeletal cell populations from heterogeneous bone marrow cell populations.

This study involves the preparation, microstructural, physical, mechanical, and biological characterization of novel gelatine and gelatine/elastin gels for their use in the tissue engineering of vascular grafts. Gelatine and gelatine/elastin nanocomposite gels were prepared via a sol-gel process, using soluble gelatine. Gelatine was subsequently cross-linked by leaving the gels in 1% glutaraldehyde. The cross-linking time was optimized by assessing the mass loss of the cross-linked gels in water and examining their mechanical properties in dynamic mechanical tests. Atomic force microscopy (AFM) studies revealed elastin nanodomains, homogeneously distributed and embedded in a bed of gelatine nanofibrils in the 30/70 elastin/gelatine gel. It was concluded that the manufactured nanocomposite gels resembled natural arteries in terms of microstructure and stiffness. The biological characterization involved the culture of rat smooth muscle cells (SMCs) on tubular gelatine and gelatine/ elastin nanocomposite gels, and measurements of the scaffold diameter and the cell density as a function of time.

Spatial patterning of cells is of great importance in tissue engineering and biotechnology, enabling, for example the creation of bottom-up histoarchitectures of heterogeneous cells, or cell aggregates for in vitro high-throughput toxicological and therapeutic studies within 3D microenvironments. In this paper, a single-step process for creating peelable and resilient hydrogels, encapsulating arrays of biological cell aggregates formed by negative DEP has been devised. The dielectrophoretic trapping within low-energy regions of the DEP-dot array reduces cell exposure to high field stresses while creating distinguishable, evenly spaced arrays of aggregates. In addition to using an optimal combination of PEG diacrylate pre-polymer solution concentration and a novel UV exposure mechanism, total processing time was reduced. With a continuous phase medium of PEG diacrylate at 15% v/v concentration, effective dielectrophoretic cell patterned arrays and photo-polymerisation of the mixture was achieved within a 4 min period. This unique single-step process was achieved using a 30 s UV exposure time frame within a dedicated, wide exposure area DEP light box system. To demonstrate the developed process, aggregates of yeast, human leukemic (K562) and HeLa cells were immobilised in an array format within the hydrogel. Relative cell viability for both cells within the hydrogels, after maintaining them in appropriate iso-osmotic media, over a week period was greater than 90%.

Temperature compensation and period determination by casein kinase 1 (CK1) are conserved features of eukaryotic circadian rhythms, whereas the clock gene transcription factors that facilitate daily gene expression rhythms differ between phylogenetic kingdoms. Human red blood cells (RBCs) exhibit temperature compensated circadian rhythms which, since RBCs lack nuclei, must occur in the absence of a circadian transcription-translation feedback loop. We tested whether period determination and temperature compensation are dependent on casein kinases in RBCs. As with nucleated cell types, broad spectrum kinase inhibition with staurosporine lengthened the period of the RBC clock at 37°C, with more specific inhibition of CK1 and CK2 also eliciting robust changes in circadian period. Strikingly, inhibition of CK1 abolished temperature compensation and increased the Q10 for the period of oscillation in RBCs, similar to observations in nucleated cells. This indicates that CK1 activity is essential for circadian rhythms irrespective of the presence or absence of clock gene expression cycles.

In this paper we show how simplifying assumptions can be used to extract useful data from the dielectrophoretic collection spectrum, in particular for the cytoplasm, and hence determine the properties of multiple populations of cells within a sample. Specifically, the observation of the frequencies of onset of dielectric dispersion allows the identification and enumeration of populations of cells according to cytoplasmic conductivity, with particular relevance to the determination of the action of drugs for high-throughput screening applications.

Most oral cancers are oral squamous cell carcinomas (OSCC) that arise from the epithelial lining of the oral mucosa. Given that the oral cavity is easily accessible, the disease lends itself to early detection; however, most oral cancers are diagnosed at a late stage, and approximately half of oral cancer sufferers do not survive beyond five years, post-diagnosis. The low survival rate has been attributed to late detection, but there is no accepted, reliable and convenient method for the detection of oral cancer and oral pre-cancer. Dielectrophoresis (DEP) is a label-free technique which can be used to obtain multi-parametric measurements of cell electrical properties. Parameters such as cytoplasmic conductivity and effective membrane capacitance (C(Eff)) can be non-invasively determined by the technique. In this study, a novel lab-on-a-chip device was used to determine the cytoplasmic conductivity and C(Eff) of primary normal oral keratinocytes, and pre-cancerous and cancerous oral keratinocyte cell lines. Our results show that the electrical properties of normal, pre-cancerous and cancerous oral keratinocytes are distinct. Furthermore, increasing C (Eff) and decreasing cytoplasmic conductivity correlate with disease progression which could prove significant for diagnostic and prognostic applications. DEP has the potential to be used as a non-invasive technique to detect oral cancer and oral pre-cancer. Clinical investigation is needed to establish the reliability and temporal relationship of the correlation between oncologic disease progression and the electrical parameters identified in this study. To use this technique as an OSCC detection tool in a clinical setting, further characterisation and refinement is warranted.

Electrical correlates of the physiological state of a cell, such as membrane conductance and capacitance, as well as cytoplasm conductivity, contain vital information about cellular function, ion transport across the membrane, and propagation of electrical signals. They are, however, difficult to measure; gold-standard techniques are typically unable to measure more than a few cells per day, making widespread adoption difficult and limiting statistical reproducibility. We have developed a dielectrophoretic platform using a disposable 3D electrode geometry that accurately (r2 > 0.99) measures mean electrical properties of populations of ~20,000 cells, by taking parallel ensemble measurements of cells at 20 frequencies up to 45 MHz, in (typically) ten seconds. This allows acquisition of ultra-high-resolution (100-point) DEP spectra in under two minutes. Data acquired from a wide range of cells – from platelets to large cardiac cells - benchmark well with patch-clamp-data. These advantages are collectively demonstrated in a longitudinal (same-animal) study of rapidly-changing phenomena such as ultradian (2–3 hour) rhythmicity in whole blood samples of the common vole (Microtus arvalis), taken from 10 µl tail-nick blood samples and avoiding sacrifice of the animal that is typically required in these studies.

It is increasingly recognised that biomimetic, natural polymers mimicking the extracellular matrix (ECM) have low thrombogenicity and functional motifs that regulate cell–matrix interactions, with these factors being critical for tissue engineered vascular grafts especially grafts of small diameter. Gelatin constitutes a low cost substitute of soluble collagen but gelatin scaffolds so far have shown generally low strength and suture retention strength. In this study, we have devised the fabrication of novel, electrospun, multilayer, gelatin fibre scaffolds, with controlled fibre layer orientation, and optimised gelatin crosslinking to achieve not only compliance equivalent to that of coronary artery but also for the first time strength of the wet tubular acellular scaffold (swollen with absorbed water) same as that of the tunica media of coronary artery in both circumferential and axial directions. Most importantly, for the first time for natural scaffolds and in particular gelatin, high suture retention strength was achieved in the range of 1.8–1.94 N for wet acellular scaffolds, same or better than that for fresh saphenous vein. The study presents the investigations to relate the electrospinning process parameters to the microstructural parameters of the scaffold, which are further related to the mechanical performance data of wet, crosslinked, electrospun scaffolds in both circumferential and axial tubular directions. The scaffolds exhibited excellent performance in human smooth muscle cell (SMC) proliferation, with SMCs seeded on the top surface adhering, elongating and aligning along the local fibres, migrating through the scaffold thickness and populating a transverse distance of 186 μm and 240 μm 9 days post-seeding for scaffolds of initial dry porosity of 74 and 83%, respectively.

Currently, cell separation occurs almost exclusively by density gradient methods and by fluorescence- and magnetic-activated cell sorting (FACS/MACS). These variously suffer from lack of specificity, high cell loss, use of labels, and high capital/operating cost. We present a dielectrophoresis (DEP)-based cell separation method, using 3D electrodes on a low-cost disposable chip; one cell type is allowed to pass through the chip whilst the other is retained and subsequently recovered. The method advances usability and throughput of DEP separation by orders of magnitude in throughput, efficiency, purity, recovery (cells arriving in the correct output fraction), cell losses (those which are unaccounted for at the end of the separation) and cost. The system was evaluated using three example separations; live and dead yeast; human cancer cells/red blood cells; and rodent fibroblasts/red blood cells. A single-pass protocol can enrich cells with cell recovery of up to 91.3% at over 300,000 cells/second with >3% cell loss. A two-pass protocol can process 300,000,000 cells in under 30 minutes, with cell recovery of up to 96.4% and cell losses below 5%, an effective processing rate >160,000 cells/second. A three-step protocol is shown to be effective for removal of 99.1% of RBCs spiked with 1% cancer cells, whilst maintaining a processing rate of ~170,000 cells/second. Furthermore, the self-contained and low-cost nature of the separator device means that it has potential application in low-contamination applications such as cell therapies, where GMP compatibility is of paramount importance. Significance statement. Cell separation is a fundamental process in biomedicine, but is presently complicated, cumbersome and expensive. We present a technique that can sort cells at a rate equivalent to or faster than gold-standard techniques such as FACS and MACs, but can do label-free and with very low cell loss. The system uses dielectrophoresis (DEP) to sort cells electrostatically, using a novel electrode chip that eschews microfabrication in favour of a laminate drilled with 397 electrode-bearing wells. This high level of parallelisation makes the system immune to the bubbles that limit labs-on-chip, whilst also increasing capacity and throughput to unprecedented levels, whilst the chip is cheap enough to be disposable, preventing inter-separation contamination.

Distinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers.

Temperature compensation and period determination by casein kinase 1 (CK1) are conserved features of eukaryotic circadian rhythms, whereas the clock gene transcription factors that facilitate daily gene expression rhythms differ between phylogenetic kingdoms. Human red blood cells (RBCs) exhibit temperature-compensated circadian rhythms, which, because RBCs lack nuclei, must occur in the absence of a circadian transcription-translation feedback loop. We tested whether period determination and temperature compensation are dependent on CKs in RBCs. As with nucleated cell types, broad-spectrum kinase inhibition with staurosporine lengthened the period of the RBC clock at 37°C, with more specific inhibition of CK1 and CK2 also eliciting robust changes in circadian period. Strikingly, inhibition of CK1 abolished temperature compensation and increased the Q10 for the period of oscillation in RBCs, similar to observations in nucleated cells. This indicates that CK1 activity is essential for circadian rhythms irrespective of the presence or absence of clock gene expression cycles.

Whilst laboratory-on-chip cell separation systems using dielectrophoresis are increasingly reported in the literature, many systems are afflicted by factors which impede "real world" performance, chief among these being cell loss (in dead spaces, attached to glass and tubing surfaces, or sedimentation from flow), and designs with large channel height-to-width ratios (large channel widths, small channel heights) that make the systems difficult to interface with other microfluidic systems. In this paper, we present a scalable structure based on 3D wells with approximately unity height-to-width ratios (based on tubes with electrodes on the sides), which is capable of enriching yeast cell populations whilst ensuring that up to 94.3% of cells processed through the device can be collected in tubes beyond the output.

Dielectrophoresis (DEP) has been used for many years for the analysis of the electrophysiological properties of cells. However, such analyses have in the past been time-consuming, such that it can take 30 min or more to collect sufficient data to make valid interpretations from a single DEP spectrum. This has limited the application of the technology to a rapid tool for non-invasive, label-free research in areas from drug discovery to diagnostics. In this paper we present the development of a programmable, multi-channel DEP system for rapid biophysical assessment of populations of biological cells. A new assay format has been developed for continuous near-real-time monitoring, using simultaneous application of up to eight alternating current electrical signals to independently addressable dot microelectrodes in an array format, allowing a DEP spectrum to be measured in 20 s, with a total cycle time between measurements of 90 s. To demonstrate the system, human leukaemic K562 cells were monitored after exposure to staurosporine and valinomycin. The DEP response curves showed the timing and manner in which the membrane properties changed for the actions of these two drugs at the early phase of induction. This technology shows the great potential for increasing our understanding of the role of electrophysiology in drug action, by observing the changes in electrical characteristics as they occur.

In cancer, multidrug resistance (MDR) is the simultaneous resistance of tumor cells to different natural product anticancer drugs that have no common structure. This is an impediment to the successful treatment of many human cancers. A common correlate of MDR is the overexpression of a membrane protein, P-glycoprotein. Many studies have shown that MDR can be reversed after the use of substrate analogs, called MDR modulators. However, our understanding of MDR modulation is incomplete. In this article, we examine the electrical properties of the human leukemic cells (K562) and its MDR counterpart (K562AR) using dielectrophoresis and flow cytometry (with a membrane potential sensitive dye, DIOC5), both before and after treatment with XR9576 (a P-glycoprotein-specific MDR-reversal agent). The results show significant differences in the cytoplasmic conductivity between the cell lines themselves, but indicate no significant changes after modulation therapy. We conclude that the process of MDR modulation is not associated with changes in the electrical properties of cancer cells. Moreover, the results demonstrate that using the flow cytometry method alone, with MDR cells, may produce artifactual results—whereas in combination with dielectrophoresis, the results show the role of MDR modulators in preventing drug efflux in MDR cells.

Electrical correlates of the physiological state of a cell, such as membrane conductance and capacitance, as well as cytoplasm conductivity, contain vital information about cellular function, ion transport across the membrane, and propagation of electrical signals. They are, however, difficult to measure; gold-standard techniques are typically unable to measure more than a few cells per day, making widespread adoption difficult and limiting statistical reproducibility. We have developed a dielectrophoretic platform using a disposable 3D electrode geometry that accurately (r2>0.99) measures mean electrical properties of populations of ~20,000 cells, by taking parallel ensemble measurements of cells at 20 frequencies up to 45 MHz, in (typically) ten seconds. This allows acquisition of ultra-high-resolution (100-point) DEP spectra in under two minutes. Data acquired from a wide range of cells – from platelets to large cardiac cells - benchmark well with patch-clamp-data. These advantages are collectively demonstrated in a longitudinal (same-animal) study of rapidly-changing phenomena such as ultradian (2-3 hour) rhythmicity in whole blood samples of the common vole (Microtus arvalis), taken from 10 µl tail-nick blood samples and avoiding sacrifice of the animal that is typically required in these studies.

OBJECTIVE: Although cells with tumorigenic/stem cell-like properties have been identified in many cancers, including oral squamous cell carcinoma (OSCC), their isolation and characterisation is still at early stages. The aim of this study is to characterise the electrophysiological properties of OSCC cells with different tumorigenic properties in order to establish if a correlation exists between tumorigenicity and cellular electrical characteristics. MATERIALS AND METHODS: Rapid adherence to collagen IV was used as a non-invasive, functional method to isolate subsets of cells with different tumorigenic abilities from one oral dysplastic and three OSCC-derived cell lines. The cell subsets identified and isolated using this method were further investigated using dielectrophoresis, a label-free method to determine their electrophysiological parameters. Cell membrane morphology was investigated using scanning electron microscopy (SEM) and modulated by use of 4-methylumbelliferone (4-MU). RESULTS: Rapid adherent cells (RAC) to collagen IV, enriched for increased tumorigenic ability, had significantly higher effective membrane capacitance than middle (MAC) and late (LAC) adherent cells. SEM showed that, in contrast to MAC and LAC, RAC displayed a rough surface, extremely rich in cellular protrusions. Treatment with 4-MU dramatically altered RAC membrane morphology by causing loss of filopodia, and significantly decreased their membrane capacitance, indicating that the highest membrane capacitance found in RAC was due to their cell membrane morphology. CONCLUSION: This is the first study showing that OSCC cells with higher tumour formation ability exhibit higher effective membrane capacitance than cells that are less tumorigenic. OSSC cells with different tumorigenic ability possessed different electrophysiological properties mostly due to their differences in the cell membrane morphology. These results suggest that dielectrophoresis could potentially used in the future for reliable, label-free isolation of putative tumorigenic cells.

Post-manufacturing thermal treatments are commonly employed in the production of hip replacements to reduce shrinkage voids which can occur in cast components. Several studies have investigated the consequences of these treatments upon the alloy microstructure and tribological properties but none have determined if there are any biological ramifications. In this study the adsorption of proteins from foetal bovine serum (FBS) on three Co-Cr-Mo ASTM-F75 alloy samples with different metallurgical histories, has been studied as a function of protein concentration. Adsorption isotherms have been plotted using the surface concentration of nitrogen as a diagnostic of protein uptake as measured by X-ray photoelectron spectroscopy. The data was a good fit to the Langmuir adsorption isotherm up to the concentration at which critical protein saturation occurred. Differences in protein adsorption on each alloy have been observed. This suggests that development of the tissue/implant interface, although similar, may differ between as-cast (AC) and heat treated samples.

Circadian rhythms organize many aspects of cell biology and physiology to a daily temporal program that depends on clock gene expression cycles in most mammalian cell types. However, circadian rhythms are also observed in isolated mammalian red blood cells (RBCs), which lack nuclei, suggesting the existence of post-translational cellular clock mechanisms in these cells. By using electrophysiological and pharmacological approaches, we show that human RBCs display circadian regulation of membrane conductance and cytoplasmic conductivity that depends on the cycling of cytoplasmic K+ levels. Using pharmacological intervention and ion replacement, we show that inhibition of K+ transport abolishes RBC electrophysiological rhythms. Our results suggest that in the absence of conventional transcription cycles, RBCs maintain a circadian rhythm in membrane electrophysiology through dynamic regulation of K+ transport.

Multilayer cellular stacks of crosslinked, electrospun 25 wt% hydroxyapatite (HA)-gelatin and pure gelatin fiber scaffolds, seeded with human foetal osteoblasts (hFOBs), were studied for up to 18 days in static and dynamic cell culture. Two types of stack models were investigated: a four-layer stack with cells seeded at the bottom surface of the first/top layer and the top surface of the fourth/bottom layer, so that the two middle layers were not seeded with cells with the aim to act as continuing conduits of culture medium and nutrients supply to the adjacent cell-populated zones; a three-layer stack with cells seeded at the bottom surface of each layer. hFOBs exhibited lower migration rate through the stack thickness for the 25 wt% HA-gelatin scaffolds compared to the pure gelatin scaffolds, due to the small pores of the former. Hence, the regularly seeded three-layer stack maintained cell-free porous zones in all layers through which the culture medium could continuously perfuse, while good fusion was achieved at the interface of all layers via the cross-migrating cells with a preference to downwards vertical migration attributed to gravity. Dynamic cell culture conditions enhanced overall cell growth by about 6% for the regularly seeded three-layer stack.

Tissue engineering of human foetal osteoblast (hFOB) cells was investigated on gelatin-hydroxyapatite (HA), crosslinked, electrospun oriented fibre scaffolds at the different hydroxyapatite concentrations of 0, 10, 20 and 25 wt% in the dry fibres and different fibre diameter, pore size and porosity of scaffolds. Rheological tests and proton NMR spectroscopy were conducted for all solutions used for electrospinning. It was found that 25 wt% HA-gelatin scaffolds electrospun at 20 kV led to the greatest cell attachment, cell proliferation and extracellular matrix (ECM) production while fibre orientation improved the mechanical properties, where crosslinked electrospun 25 wt% HA-gelatin fibre scaffolds yielded a Young’s modulus in the range of 0.5 to 0.9 GPa and a tensile strength in the range of 4 to 10 MPa in the fibre direction for an applied voltage of 20 to 30 kV, respectively, in the electrospinning of scaffolds. Biological characterisation of cell seeded scaffolds yielded the rate of cell growth and ECM (collagen and calcium) production by the cells as a function of time; it included cell seeding efficiency tests, alamar blue cell proliferation assay, alkaline phosphate (ALP) assay, collagen assay, calcium colorimetric assay, fluorescence microscopy for live and dead cells, and scanning electron microscopy (SEM) for cell culture from 1 to 18 days. After 18 days, cells seeded and grown on the 25 wt% HA-gelatin scaffold, electrospun at 20 kV, reached production of collagen at 370 g/L and calcium production at 0.8 mM.

Whilst personalized medicine (where interventions are precisely tailored to a patient’s genotype and phenotype, as well as the nature and state of the disease) is regarded as an optimal form of treatment, the time and cost associated with it means it remains inaccessible to the greater public. A simpler alternative, stratified medicine, identifies groups of patients who are likely to respond to a given treatment. This allows appropriate treatments to be selected at the start of therapy, avoiding the common “trial and error” approach of replacing a therapy only once it is demonstrated to be ineffective in the patient. For stratification to be effective, tests are required that rapidly predict treatment effectiveness. Most tests use genetic analysis to identify drug targets, but these can be expensive and may not detect changes in the phenotype that affect drug sensitivity. An alternative method is to assess the whole-cell phenotype by evaluating drug response using cells from a biopsy. We assessed dielectrophoresis to assess drug efficacy on short timescales and at low cost. To explore the principle of assessing drug efficacy we examined two cell lines (one expressing EGFR, one not) with the drug Iressa. We then further explored the sensitive cells using combinations of chemotherapeutic and radiotherapeutic therapies. Our results compare with known effects of these cell/treatment combination, and offer the additional benefit over methods such as TUNEL of detecting drug effects such as cell cycle arrest, which do not cause cell death.

BACKGROUND: Many drug development and toxicology studies are performed using cells grown in monolayers in well-plates and flasks, despite the fact that these are widely held to be different to cells found in the native environment. 3D, tissue engineered, organotypical tissue culture systems have been developed to be more representative of the native tissue environment than standard monolayer cultures. Whilst the biochemical differences between cells grown in 2D and 3D culture have been explored, the changes on the electrophysiological properties of the cells have not. METHODS: We compared the electrophysiological properties of primary normal oral keratinocytes (nOK) and cancerous abnormal oral keratinocytes (aOK), cultured in standard monolayer and reconstituted 3D organotypical tissue cultures. The electrophysiological properties of populations of the cells were analysed using dielectrophoresis. The intracellular conductivity of aOK was significantly increased when grown in organotypical cultures compared to counterpart cells grown in monolayer cultures. RESULTS: 3D cultured aOK showed almost identical intracellular conductivity to nOK also grown in organotypical cultures, but significantly different to aOK grown in monolayers. The effective membrane capacitance of aOK grown in 3D was found to be significantly higher than nOK, but there was no significant difference between the electrophysiological properties of nOK grown in 2D and 3D cultures. GENERAL SIGNIFICANCE: This work suggests that factors such as cell shape and cytoplasmic trafficking between cells play an important role in their electrophysiology, and highlights the need to use in vitro models more representative of native tissue when studying cell electrophysiological properties.

It is known that cells grown in 3D are more tolerant to drug treatment than those grown in dispersion but the mechanism for this is still not clear; cells grown in 3D have opportunities to develop inter-cell communication, but are also closely packed which may impede diffusion. In this study we examine methods for dielectrophoresis-based cell aggregation of both suspension and adherent cell lines and compare the effect of various drugs on cells grown in 3D and 2D. Comparing viability of pharmacological interventions on 3D cell clusters against both suspension cells and adherent cells grown in monolayer, as well as against a unicellular organism with no propensity for intracellular communication, we suggest that 3D aggregates of adherent cells, compared to suspension cells, show a substantially different drug response to cells grown in monolayer, which increases as the IC50 is approached. Further, a mathematical model of the system for each agent demonstrates that changes to drug response are due to inherent changes in the system of adherent cells from the 2D to 3D state. Finally, differences within electrophysiological membrane properties of the adherent cell type suggest this parameter plays an important role in the differences found in the 3D drug response.

Cellular circadian rhythms confer temporal organisation upon physiology that is fundamental to human health. Rhythms are present in red blood cells (RBCs), the most abundant cell type in the body, but their physiological function is poorly understood. Here, we present a novel biochemical assay for haemoglobin (Hb) oxidation status which relies on a redox-sensitive covalent haem-Hb linkage that forms during SDS-mediated cell lysis. Formation of this linkage is lowest when ferrous Hb is oxidised, in the form of ferric metHb. Daily haemoglobin oxidation rhythms are observed in mouse and human RBCs cultured in vitro, or taken from humans in vivo, and are unaffected by mutations that affect circadian rhythms in nucleated cells. These rhythms correlate with daily rhythms in core body temperature, with temperature lowest when metHb levels are highest. Raising metHb levels with dietary sodium nitrite can further decrease daytime core body temperature in mice via nitric oxide (NO) signalling. These results extend our molecular understanding of RBC circadian rhythms and suggest they contribute to the regulation of body temperature.

Diseases such as osteoarthritis (OA) are commonly characterized at the molecular scale by gene expression and subsequent protein production; likewise, the effects of pharmaceutical interventions are typically characterized by the effects of molecular interactions. However, these phenomena are usually preceded by numerous precursor steps, many of which involve significant ion influx or efflux. As a consequence, rapid assessment of cell electrophysiology could play a significant role in unravelling the mechanisms underlying drug interactions and progression of diseases, such as OA. In this study, we used dielectrophoresis (DEP), a technique that allows rapid, label-free determination of the dielectric parameters to assess the role of potassium ions on the dielectric characteristics of chondrocytes, and to investigate the electrophysiological differences between healthy chondrocytes and those from an in vitro arthritic disease model. Our results showed that DEP was able to detect a significant decrease in membrane conductance (6191 ± 738 vs. 8571 ± 1010 S/m2), membrane capacitance (10.3 ± 1.47 vs. 14.5 ± 0.01 mF/m2), and whole cell capacitance (5.4 ± 0.7 vs. 7.5 ± 0.3 pF) following inhibition of potassium channels using 10 mM tetraethyl ammonium, compared to untreated healthy chondrocytes. Moreover, cells from the OA model had a different response to DEP force in comparison to healthy cells; this was seen in terms of both a decreased membrane conductivity (782 S/m2 vs. 1139 S/m2) and a higher whole cell capacitance (9.58 ± 3.4 vs. 3.7 ± 1.3 pF). The results show that DEP offers a high throughput method, capable of detecting changes in membrane electrophysiological properties and differences between disease states.

Chondrogenic progenitor cells (CPCs) may be used as an alternative source of cells with potentially superior chondrogenic potential compared to mesenchymal stem cells (MSCs), and could be exploited for future regenerative therapies targeting articular cartilage in degenerative diseases such as osteoarthritis (OA). In this study, we hypothesised that CPCs derived from OA cartilage may be characterised by a distinct channelome. First, a global transcriptomic analysis using Affymetrix microarrays was performed. We studied the profiles of those ion channels and transporter families that may be relevant to chondroprogenitor cell physiology. Following validation of the microarray data with quantitative reverse transcription-polymerase chain reaction, we examined the role of calcium-dependent potassium channels in CPCs and observed functional large-conductance calcium-activated potassium (BK) channels involved in the maintenance of the chondroprogenitor phenotype. In line with our very recent results, we found that the KCNMA1 gene was upregulated in CPCs and observed currents that could be attributed to the BK channel. The BK channel inhibitor paxilline significantly inhibited proliferation, increased the expression of the osteogenic transcription factor RUNX2, enhanced the migration parameters, and completely abolished spontaneous Ca events in CPCs. Through characterisation of their channelome we demonstrate that CPCs are a distinct cell population but are highly similar to MSCs in many respects. This study adds key mechanistic data to the in-depth characterisation of CPCs and their phenotype in the context of cartilage regeneration.

Introduction. Platelets protect the body from injury through formation of blood clots, changing from a normal, quiescent state to become “activated” in response to external stimuli such as chemical cues, shear stress and temperature. This causes changes in shape, increased adhesion, and alteration of the electrical properties such as membrane potential Vm and zeta potential ζ. These phenomena have been regarded as largely unconnected; for example, changes in ζ have been attributed solely to alteration of surface lipid concentration. However, recent reports suggest that cells can alter ζ electrostatically by alteration of Vm in red blood cells. We hypothesised that if platelets also modulate ζ via Vm, this may provide an alternative mechanism to alter cell-cell interaction. Methods. We investigated platelets stored at different temperatures (4°C, 22°C, 37°C) for 24h, which is known to alter platelet behaviour and electrical properties, and compared these with analyses of freshly-harvested platelets. These four conditions exhibited unique sets of electrical properties (Vm, ζ, membrane conductance Geff and cytoplasm conductivity σcyto) as well as surface exposure of the adhesion molecule P-Selectin. These were analysed to identify correlations between electrical parameters and platelet activation state. Results. Many parameters exhibit pairwise correlation across all four conditions, in particular between ζ and Geff, and also between Vm and σcyto. Furthermore, when the electrical behaviour of platelets stored at 4°C (known to activate the cells) was removed from the analysis, additional relationships were observed among the remaining conditions, including those connecting ζ and Vm to the amount of P-selectin binding. Conclusion. Results suggest that Vm may mechanistically alter the availability of cationic molecules at the cell surface, a process never reported before, with implications for our wider understanding of cell-molecule and cell-cell interaction.

Dielectrophoresis is an electrical phenomenon that occurs when a polarisable particle is placed in non-uniform electrical fields. The magnitude of the generated force is dependent upon the electrophysiological make-up of the particle, therefore the specific DEP profile may be attained for any polarisable particles based on the intrinsic electrical properties alone. Any changes to these parameters may be detected by observing the corresponding DEP spectra. Despite having the advantages of being non-invasive, DEP applications are still not widely used due to the time-consuming processes involved. This study presents the preliminary outcomes in the development of a semi-automated DEP-based cell characterisation tool that allowed concurrent DEP experiments to be conducted serially, thus significantly reducing the time taken to complete the required sets of experiments. The results showed that the system is capable of producing a DEP spectrum for K562 leukaemic cells between the 10 kHz to 1 MHz range in less than 10 minutes, when recorded at eight points per decade.

In this paper, the use of non-uniform ac electric fields on biological cells for bioanalysis, through multiple, independently configurable channels is presented. The programmable system has been used to obtain the dielectrophoretic spectra of cells in near real time, within 90 seconds. This is a significant improvement on existing dielectrophoretic techniques as simultaneous parallel measurement of the dielectrophoretic forces at different frequencies has potential of revealing subtle changes to the electrophysiology of cells, as they occur. The results show that with continuous on-chip monitoring, cells exposed to a chemical agent that induces apoptosis begin to exhibit a spectrum that differs from untreated cells, as indicated from shifts in the observed crossover frequency values.

Dielectrophoresis (DEP) is a phenomenon of induced particle motion in non-uniform electric fields. The effect is frequency dependent; by monitoring the motion of particles in AC fields and analyzing the change in motion with frequency, it is possible to determine the electrical properties of single cells in lab-on-a-chip systems. By combining two common electrokinetic phenomena dielectrophoresis and electrohydrodynamic fluid flow - we demonstrate that it is possible to manipulate, concentrate and trap particles from cell to molecular scale, and show how the trapping phenomenon is not related to particle size. We also discuss application of the phenomenon, from particle preconcentration in sensor systems to the deposition of particles on sensor surfaces.

In this article, which is related to the biomedical signal processing applications area of the workshop, we present a system for positioning a single-neuron inside each micro-well of a 4-by-4 planar microelectrode array (MEA). Neurons are moved toward the electrode sites of the MEA (located at the bottom of the wells) using dielectrophoresis. The system utilizes the image acquisition and processing capabilities of MATLAB to detect the presence of a neuron inside each micro-well and stop the dielectrophoretic force, thus preventing more cells from being loaded. This method provides a fast, simple and relatively inexpensive way for loading cells on MEAs embedded with micro-wells for the purpose of acquiring and processing action potentials from geometrically defined biological neural networks at the single-cell level. Recordings from neurons that were positioned using this system have been obtained and are presented. © 2013 IEEE.

Dielectrophoresis (DEP) is a phenomenon of induced particle motion in non-uniform electric fields. The effect is frequency dependent; by monitoring the motion of particles in AC fields and analysing the change in motion with frequency, it is possible to determine the electrical properties of single cells in lab-on-a-chip systems. In this paper we use DEP to study the electrical properties of drug-sensitive and drug-resistant cancer cells before and after treatment with ion channel blocking agents, which give insight into the origin of cytoplasmic differences shown to have a significant bearing on the origin of drug resistance in cancer.

In this paper, the use of non-uniform ac electric fields on biological cells for bioanalysis, through multiple, independently configurable channels is presented. The programmable system has been used to obtain the dielectrophoretic spectra of cells in near real time, within 90 seconds. This is a significant improvement on existing dielectrophoretic techniques as simultaneous parallel measurement of the dielectrophoretic forces at different frequencies has potential of revealing subtle changes to the electrophysiology of cells, as they occur. The results show that with continuous on-chip monitoring, cells exposed to a chemical agent that induces apoptosis begin to exhibit a spectrum that differs from untreated cells, as indicated from shifts in the observed crossover frequency values.

This article succeeded to introduce a new method, that mimics the radio broadcasting, used amplitude modulation to deliver the low range frequencies to the cell membrane. Consequently, avoiding the ACEO which prevents the measurement of DEP spectrum. In addition to the use of quinine and NBBP as ion channel blockers along with the modulated dielectrophoresis technique enabled the measurement of membrane conductivity providing a low cost method. © 2013 Trade Science Inc.-INDIA.