Trajectories and Forces in Four-Electrode Chambers Operated in Object-Shift, Dielectrophoresis and Field-Cage Modes-Considerations from the System's Point of View.

In two previous papers, we calculated the dielectrophoresis (DEP) force and corresponding trajectories of high- and low-conductance 200-µm 2D spheres in a square 1 × 1-mm chamber with plane-versus-pointed, plane-versus-plane and pointed-versus-pointed electrode configurations by applying the law of maximum entropy production (LMEP) to the system. Here, we complete these considerations for configurations with four-pointed electrodes centered on the chamber edges. The four electrodes were operated in either object-shift mode (two adjacent electrodes opposite the other two adjacent electrodes), DEP mode (one electrode versus the other three electrodes), or field-cage mode (two electrodes on opposite edges versus the two electrodes on the other two opposite edges). As in previous work, we have assumed DC properties for the object and the external media for simplicity. Nevertheless, every possible polarization ratio of the two media can be modeled this way. The trajectories of the spherical centers and the corresponding DEP forces were calculated from the gradients of the system's total energy dissipation, described by numerically-derived conductance fields. In each of the three drive modes, very high attractive and repulsive forces were found in front of pointed electrodes for the high and low-conductance spheres, respectively. The conductance fields predict bifurcation points, watersheds, and trajectories with multiple endpoints. The high and low-conductance spheres usually follow similar trajectories, albeit with reversed orientations. In DEP drive mode, the four-point electrode chamber provides a similar area for DEP measurements as the classical plane-versus-pointed electrode chamber.

The System's Point of View Applied to Dielectrophoresis in Plate Capacitor and Pointed-versus-Pointed Electrode Chambers.

The DEP force is usually calculated from the object's point of view using the interaction of the object's induced dipole moment with the inducing field. Recently, we described the DEP behavior of high- and low-conductive 200-µm 2D spheres in a square 1 × 1-mm chamber with a plane-versus-pointed electrode configuration from the system's point of view. Here we extend our previous considerations to the plane-versus-plane and pointed-versus-pointed electrode configurations. The trajectories of the sphere center and the corresponding DEP forces were calculated from the gradient of the system's overall energy dissipation for given starting points. The dissipation's dependence on the sphere's position in the chamber is described by the numerical "conductance field", which is the DC equivalent of the capacitive charge-work field. While the plane-versus-plane electrode configuration is field-gradient free without an object, the presence of the highly or low-conductive spheres generates structures in the conductance fields, which result in very similar DEP trajectories. For both electrode configurations, the model describes trajectories with multiple endpoints, watersheds, and saddle points, very high attractive and repulsive forces in front of pointed electrodes, and the effect of mirror charges. Because the model accounts for inhomogeneous objectpolarization by inhomogeneous external fields, the approach allows the modeling of the complicated interplay of attractive and repulsive forces near electrode surfaces and chamber edges. Non-reversible DEP forces or asymmetric magnitudes for the highly and low-conductive spheres in large areas of the chamber indicate the presence of higher-order moments, mirror charges, etc.

Dielectrophoresis from the System's Point of View: A Tale of Inhomogeneous Object Polarization, Mirror Charges, High Repelling and Snap-to-Surface Forces and Complex Trajectories Featuring Bifurcation Points and Watersheds.

Microscopic objects change the apparent permittivity and conductivity of aqueous systems and thus their overall polarizability. In inhomogeneous fields, dielectrophoresis (DEP) increases the overall polarizability of the system by moving more highly polarizable objects or media to locations with a higher field. The DEP force is usually calculated from the object's point of view using the interaction of the object's induced dipole or multipole moments with the inducing field. Recently, we were able to derive the DEP force from the work required to charge suspension volumes with a single object moving in an inhomogeneous field. The capacitance of the volumes was described using Maxwell−Wagner's mixing equation. Here, we generalize this system's-point-of-view approach describing the overall polarizability of the whole DEP system as a function of the position of the object with a numerical "conductance field". As an example, we consider high- and low conductive 200 µm 2D spheres in a square 1 × 1 mm chamber with plain-versus-pointed electrode configuration. For given starting points, the trajectories of the sphere and the corresponding DEP forces were calculated from the conductance gradients. The model describes watersheds; saddle points; attractive and repulsive forces in front of the pointed electrode, increased by factors >600 compared to forces in the chamber volume where the classical dipole approach remains applicable; and DEP motions with and against the field gradient under "positive DEP" conditions. We believe that our approach can explain experimental findings such as the accumulation of viruses and proteins, where the dipole approach cannot account for sufficiently high holding forces to defeat Brownian motion.

Contributions to a Discussion of Spinosaurus aegyptiacus as a Capable Swimmer and Deep-Water Predator.

The new findings on Spinosaurus' swim tail strongly suggest that Spinosaurus was a specialized deep-water predator. However, the tail must be seen in the context of the propelled body. The comparison of the flow characteristics of Spinosaurus with geometrically similar animals and their swimming abilities under water must take their Reynolds numbers into account and provide a common context for the properties of Spinosaurus' tail and dorsal sail. Head shape adaptations such as the head crest reduced hydrodynamic disturbance and facilitated stealthy advance, especially when hunting without visual contact, when Spinosaurus could have used its rostral integumentary mechanoreceptors for prey detection. The muscular neck permitted 'pivot' feeding, where the prey's escape abilities were overcome by rapid dorsoventral head movement, facilitated by crest-mediated lower friction.

Active, Reactive, and Apparent Power in Dielectrophoresis: Force Corrections from the Capacitive Charging Work on Suspensions Described by Maxwell-Wagner's Mixing Equation.

A new expression for the dielectrophoresis (DEP) force is derived from the electrical work in a charge-cycle model that allows the field-free transition of a single object between the centers of two adjacent cubic volumes in an inhomogeneous field. The charging work for the capacities of the volumes is calculated in the absence and in the presence of the object using the external permittivity and Maxwell-Wagner's mixing equation, respectively. The model provides additional terms for the Clausius-Mossotti factor, which vanish for the mathematical boundary transition toward zero volume fraction, but which can be interesting for narrow microfluidic systems. The comparison with the classical solution provides a new perspective on the notorious problem of electrostatic modeling of AC electrokinetic effects in lossy media and gives insight into the relationships between active, reactive, and apparent power in DEP force generation. DEP moves more highly polarizable media to locations with a higher field, making a DEP-related increase in the overall polarizability of suspensions intuitive. Calculations of the passage of single objects through a chain of cubic volumes show increased overall effective polarizability in the system for both positive and negative DEP. Therefore, it is proposed that DEP be considered a conditioned polarization mechanism, even if it is slow with respect to the field oscillation. The DEP-induced changes in permittivity and conductivity describe the increase in the overall energy dissipation in the DEP systems consistent with the law of maximum entropy production. Thermodynamics can help explain DEP accumulation of small objects below the limits of Brownian motion.

Subthalamic nucleus deep brain stimulation induces sustained neurorestoration in the mesolimbic dopaminergic system in a Parkinson's disease model.

BACKGROUND: Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an established therapeutic principle in Parkinson's disease, but the underlying mechanisms, particularly mediating non-motor actions, remain largely enigmatic. OBJECTIVE/HYPOTHESIS: The delayed onset of neuropsychiatric actions in conjunction with first experimental evidence that STN-DBS causes disease-modifying effects prompted our investigation on how cellular plasticity in midbrain dopaminergic systems is affected by STN-DBS. METHODS: We applied unilateral or bilateral STN-DBS in two independent cohorts of 6-hydroxydopamine hemiparkinsonian rats four to eight weeks after dopaminergic lesioning to allow for the development of a stable dopaminergic dysfunction prior to DBS electrode implantation. RESULTS: After 5 weeks of STN-DBS, stimulated animals had significantly more TH+ dopaminergic neurons and fibres in both the nigrostriatal and the mesolimbic systems compared to sham controls with large effect sizes of gHedges = 1.9-3.4. DBS of the entopeduncular nucleus as the homologue of the human Globus pallidus internus did not alter the dopaminergic systems. STN-DBS effects on mesolimbic dopaminergic neurons were largely confirmed in an independent animal cohort with unilateral STN stimulation for 6 weeks or for 3 weeks followed by a 3 weeks washout period. The latter subgroup even demonstrated persistent mesolimbic dopaminergic plasticity after washout. Pilot behavioural testing showed that augmentative dopaminergic effects on the mesolimbic system by STN-DBS might translate into improvement of sensorimotor neglect. CONCLUSIONS: Our data support sustained neurorestorative effects of STN-DBS not only in the nigrostriatal but also in the mesolimbic system as a potential factor mediating long-latency neuropsychiatric effects of STN-DBS in Parkinson's disease.

A comparative analysis of detachment forces and energies in initial and mature cell-material interaction.

Single cell force spectroscopy (SCFS) enables data on interaction forces to be acquired during the very early adhesion phase. However, SCFS detachment forces and energies have not been compared so far with the forces and energies after maturation of the cell-material contact on a single cell level and with comparable time resolution. We used FluidFM® to physically attach single cells to the cantilever by aspiration through a microfluidic channel, in order to achieve the higher forces required for detaching maturely adhering cells. Combining these two approaches allowed us to compare cell adhesion in the initial and maturation phases of adhesion for two exemplary cell-substrate combinations - L929 fibroblasts on fibronectin and MC3T3 osteoblasts on collagen type I. Uncoated glass substrates were used as a reference. For both cell lines, SCFS measurements after contact times of 5, 15 and 30 s revealed significantly higher maximum detachment forces (MDFs) and energies on glass compared to the protein-coated surfaces in the 0.5-4 nN (1-40 fJ) range. FluidFM® measurements after 1, 2 and 3 days of culture revealed a significant absolute increase in the MDFs and detachment energies for both cell lines on protein-coated substrates to values of about 600 nN and 10 pJ. On glass, the MDFs were similar for MC3T3 cells, while they were significantly lower for L929 cells. For both cell types, the differences in detachment energy were significant. These differences underline the importance of investigating early and mature adhesion states to obtain a holistic assessment of the cell-material interactions.

Surface Coatings Modulate the Differences in the Adhesion Forces of Eukaryotic and Prokaryotic Cells as Detected by Single Cell Force Microscopy.

Single cell force microscopy was used to investigate the maximum detachment force (MDF) of primary neuronal mouse cells (PNCs), osteoblastic cells (MC3T3), and prokaryotic cells (Staphylococcus capitis subsp. capitis) from different surfaces after contact times of 1 to 5 seconds. Positively charged silicon nitride surfaces were coated with positively charged polyethyleneimine (PEI) or poly-D-lysine. Laminin was used as the second coating. PEI induced MDFs of the order of 5 to 20 nN, slightly higher than silicon nitride did. Lower MDFs (1 to 5 nN) were detected on PEI/laminin with the lowest on PDL/laminin. To abstract from the individual cell properties, such as size, and to obtain cell type-specific MDFs, the MDFs of each cell on the different coatings were normalized to the silicon nitride reference for the longest contact time. The differences in MDF between prokaryotic and eukaryotic cells were generally of similar dimensions, except on PDL/laminin, which discriminated against the prokaryotic cells. We explain the lower MDFs on laminin by the spatial prevention of the electrostatic cell adhesion to the underlying polymers. However, PEI can form long flexible loops protruding from the surface-bound layer that may span the laminin layer and easily bind to cellular surfaces and the small prokaryotic cells. This was reflected in increased MDFs after two-second contact times on silicon nitride, whereas the two-second values were already observed after one second on PEI or PEI/laminin. We assume that the electrostatic charge interaction with the PEI loops is more important for the initial adhesion of the smaller prokaryotic cells than for eukaryotic cells.

Combined detection of AC-electrokinetic effects: Experiments with three-axial chicken red blood cells.

Dielectrophoresis (DEP), electrorotation (ROT), and electro-orientation were used for the dielectric spectroscopy of nucleated three-axial chicken red blood cells (CRBCs). Because the different AC-electrokinetic effects are not mutually independent, their DEP and ROT spectra were combined in ranges separated by the reorientation of the CRBCs in the inhomogeneous linear DEP and circular ROT fields. This behavior can be qualitatively described by a single-shell ellipsoidal model. Whereas in linear fields, the maximum of the Clausius-Mossotti factor along the three axes determines the orientated axis, in circular fields, the minimum of the factor determines the axis perpendicularly orientated to the field plane. Quantitatively, it has not been possible to find a consistent parameter set for fitting the DEP and ROT spectra, as well as the reorientation frequencies. Our ellipsoidal CRBC standard model had semiaxes of a = 7.7 µm, b = 4.0 µm, and c = 1.85 µm, a relative permittivity of 35 to 45 and conductivity of 0.36 to 0.04 S/m for the cytoplasm, combined with a specific capacitance of 10 to 14 mF/m2 and a conductivity of 3500 S/m2 for the cell membrane. The fits in different external conductivity ranges between external conductivities of 0.015 and 1.0 S/m were improved when the membrane capacitance was changed between 4 to 25 mF/m2 depending on the method used. A similar transition was reflected in the effective properties of a three-shell spherical model containing an internal membranous sphere with the geometry of the CRBC nucleus. Our findings suggest that the simultaneous interpretation of various AC-electrokinetic spectra is a step toward the dielectric fingerprinting of biological cells.

Combined AC-electrokinetic effects: Theoretical considerations on a three-axial ellipsoidal model.

AC fields induce charges at the structural interfaces of particles or biological cells. The interaction of these charges with the field generates frequency-dependent forces that are the basis for AC-electrokinetic effects such as dielectrophoresis (DEP), electrorotation (ROT), electro-orientation, and electro-deformation. The effects can be used for the manipulation or dielectric single-particle spectroscopy. The observation of a particular effect depends on the spatial and temporal field distributions, as well as on the shape and the dielectric and viscoelastic properties of the object. Because the effects are not mutually independent, combined frequency spectra are obtained, for example, discontinuous DEP and ROT spectra with ranges separated by the reorientation of nonspherical objects in the linearly and circularly polarized DEP and ROT fields, respectively. As an example, the AC electrokinetic behavior of a three-axial ellipsoidal single-shell model with the geometry of chicken-red blood cells is considered. The geometric and electric problems were separated using the influential-radius approach. The obtained finite-element model can be electrically interpreted by an RC model leading to an expression for the Clausius-Mossotti factor, which permits the derivation of force, torque, and orientation spectra, as well as of equations for the critical frequencies and force plateaus in DEP and of the characteristic frequencies and peak heights in ROT. Expressions for the orientation in linearly and circularly polarized fields, as well as for the reorientation frequencies were also derived. The considerations suggested that the simultaneous registration of various AC-electrokinetic spectra is a step towards the dielectric fingerprinting of single objects.

Spermidine-Induced Attraction of Like-Charged Surfaces Is Correlated with the pH-Dependent Spermidine Charge: Force Spectroscopy Characterization.

The ubiquitous molecule spermidine is known for its pivotal roles in the contact mediation, fusion, and reorganization of biological membranes and DNA. In our model system, borosilicate beads were attached to atomic force microscopy cantilevers and used to probe mica surfaces to study the details of the spermidine-induced attractions. The negative surface charges of both materials were largely constant over the measured pH range of pH 7.8 to 12. The repulsion observed between the surfaces turned into attraction after the addition of spermidine. The attractive force was correlated with the degree of spermidine protonation, which changed from +3 to +1 over the measured pH range. The force was maximal at pH 7.8. To explain the observed pH and spermidine concentration dependence, two different theoretical approaches were used: a chemical model of the charge equilibrium of spermidine and Monte-Carlo simulations of the orientation of the rodlike spermidine molecules in the gap between the borosilicate and mica surfaces. Monte-Carlo simulations of the orientational ordering of the rodlike spermidine molecules suggested the induction of attractive interactions between the surfaces if the gap was bridged by the molecules. For larger gaps, the orientational distribution function of the spermidine molecules predicted a considerable degree of parallel attachment of the molecules to the surfaces, resulting in reduced effective surface charge densities of both surfaces, which reduced their electrostatic repulsion.

Furthering the state of knowledge on the electric properties of hemi-ellipsoidal single cells and cell patches on electrodes.

The impedance of electrodes with adherent biological cells correlates with cell viability and proliferation. To model this correlation, we exploited the idea that the introduction of a highly conductive layer into the equatorial equipotential slice of a system with an oriented, freely suspended, single ellipsoidal cell may split the system into mirror-symmetrical halves without changing the field distribution. Each half possesses half of the system's impedance and contains a hemiellipsoidal cell attached to the conductive layer, which can be considered a bottom electrode. For a hemiellipsoidal adherent cell model (ACM) with standard electrical properties for the external and cellular media, the assumption of a bottom membrane and a subcellular cleft in the 100 nm range, as found in adherent cells, changed the potential distribution over a one-% range up to frequencies of 1 MHz. For simplicity, potential distributions for slices of spheroidal objects can be numerically calculated in 2D. The 2D distributions can be converted into three dimensions using simplified equations for the influential radii of spheroids. After the ACM approach was expanded to adherent cell patch models (APMs), the feasibility of our model modifications was tested using two criteria: the constancy of the equipotential plane touching the poles of ACMs or APMs and a comparison of the impedance, which could be numerically calculated from the overall current between the bottom electrode and a plane-parallel counter-electrode, with the impedance of the suspension obtained from Maxwell-Wagner's mixing equation applied to hemiellipsoidal cells.

Increased osteoblast viability at alkaline pH in vitro provides a new perspective on bone regeneration.

We investigated the effects of alkaline pH on developing osteoblasts. Cells of the osteoblast-like cell line MC3T3-E1 were initially cultured for six days in HEPES-buffered media with pH ranging from 7.2 to 9.0. Cell count, cellular WST-1 metabolism, and ATP content were analyzed. The three parameters showed a pH optimum around pH 8.4, exceeding the recommended buffer range of HEPES at the alkaline flank. Therefore, only pH 7.2, 7.4, 7.8, and 8.4 media were used in more elaborate, daily investigations to reduce the effects of pH change within the pH control intervals of 24 h. All parameters exhibited similar pH behaviors, roughly showing increases to 130% and 230% at pH 7.8 and 8.4, as well as decreases to 70% at pH 7.2 when using the pH 7.4 data for reference. To characterize cell differentiation and osteoblastic cell function, cells were cultured at pH 7.4 and under alkaline conditions at pH 7.8 and 8.4 for 14 days. Gene expression and mineralization were evaluated using microarray technology and Alizarin staining. Under alkaline conditions, ATF4, a regulator for terminal differentiation and function as well as DMP1, a potential marker for the transition of osteoblasts into osteocytes, were significantly upregulated, hinting at an accelerated differentiation process. After 21 days, significant mineralization was only detected at alkaline pH. We conclude that elevated pH is beneficial for the cultivation of bone cells and may also provide therapeutic value in bone regeneration therapies.

Deep Brain Stimulation of Hemiparkinsonian Rats with Unipolar and Bipolar Electrodes for up to 6 Weeks: Behavioral Testing of Freely Moving Animals.

Although the clinical use of deep brain stimulation (DBS) is increasing, its basic mechanisms of action are still poorly understood. Platinum/iridium electrodes were inserted into the subthalamic nucleus of rats with unilateral 6-OHDA-induced lesions of the medial forebrain bundle. Six behavioral parameters were compared with respect to their potential to detect DBS effects. Locomotor function was quantified by (i) apomorphine-induced rotation, (ii) initiation time, (iii) the number of adjusting steps in the stepping test, and (iv) the total migration distance in the open field test. Sensorimotor neglect and anxiety were quantified by (v) the retrieval bias in the corridor test and (vi) the ratio of migration distance in the center versus in the periphery in the open field test, respectively. In our setup, unipolar stimulation was found to be more efficient than bipolar stimulation for achieving beneficial long-term DBS effects. Performance in the apomorphine-induced rotation test showed no improvement after 6 weeks. DBS reduced the initiation time of the contralateral paw in the stepping test after 3 weeks of DBS followed by 3 weeks without DBS. Similarly, sensorimotor neglect was improved. The latter two parameters were found to be most appropriate for judging therapeutic DBS effects.

MC3T3 osteoblast-like cells cultured at alkaline pH: Microarray data (Affymetrix GeneChip Mouse 2.0 ST).

It is well known that pH plays a pivotal role in the control of bone remodeling. However, no comprehensive gene expression data are available for the effects of alkaline pH on osteoblasts. We cultured differentiating MC3T3-E1 osteoblast-like cells at pH 7.4, 7.8, and 8.4 for 14 days. To identify differential gene expression, microarray data were collected with Affymetrix GeneChips. The data were validated by real-time PCRs for five genes that were found to be greatly regulated in the GeneChip-experiments (DMP1, FABP4, SFRP2 and TNFRSF19) or considered relevant for the terminal function of osteoblasts (DMP1 and ATF4). All the data are available from the Gene Expression Omnibus database (GEO accession: GSE84907). Here, we provide pathway analyses of known protein coding genes that were down-regulated or up-regulated by greater than 2.0-fold. The regulation datasets obtained from comparisons of pH 7.8 and 7.4, as well as pH 8.4 and 7.4 share a high number of differentially expressed genes. When comparing pH 8.4 and 7.8, other genes mainly emerge, suggesting not only a simple amplification of the effects at pH 8.4 that were already induced at pH 7.8 but also the induction of different pathways. For a more detailed analysis, different mammalian functional gene networks were assigned to each dataset. After merging and manual optimization of the network graphs, three combined functional gene networks were obtained that reflected distinct pH-dependent cellular responses. A common feature of the networks was the central role of p38 MAP kinase. The microarray data presented here are related to the research article doi:10.1016/j.bbrep.2017.02.001 (Galow et al., 2017) [1].

WST-assay data reveal a pH dependence of the mitochondrial succinate reductase in osteoblast-like cells.

The data presented in this article are related to the research article entitled "Increased osteoblast viability at alkaline pH in vitro provides a new perspective on bone regeneration" (doi: 10.1016/j.bbrep.2017.02.001; (Galow et al., 2017) [1]). The water soluble tetrazolium (WST) proliferation assay detects the metabolic activity of the respiratory chain of cultured cells. The assay is based on changes in the light absorbance resulting from the metabolism of WST-1 into formazane by mitochondrial succinate reductase. We present data of three different tests that were carried out to check whether WST assay readouts are pH-dependent. In a first test, a possible pH effect on the photometric measurements, for example by shifting the absorbance spectrum of the pH indicator of the cell culture medium, was excluded. Because the second test revealed a significant pH-dependence of the activity of the mitochondrial succinate reductase, a third long-term test was conducted to analyze possible changes of the pH dependence over time. The higher absorbance per one million cells at alkaline pH, which was approximately four-fold at pH 8.4 compared to the pH-7.4 reference on day one decayed gradually, with the pH-differences equilibrating over six days.

Automated and manual patch clamp data of human induced pluripotent stem cell-derived dopaminergic neurons.

Human induced pluripotent stem cells can be differentiated into dopaminergic neurons (Dopa.4U). Dopa.4U neurons expressed voltage-gated NaV and KV channels and showed neuron-like spontaneous electrical activity. In automated patch clamp measurements with suspended Dopa.4U neurons, delayed rectifier K+ current (delayed KV) and rapidly inactivating A-type K+ current (fast KV) were identified. Examination of the fast KV current with inhibitors yielded IC50 values of 0.4 mM (4-aminopyridine) and 0.1 mM (tetraethylammonium). In manual patch clamp measurements with adherent Dopa.4U neurons, fast KV current could not be detected, while the delayed KV current showed an IC50 of 2 mM for 4-aminopyridine. The NaV channels in adherent and suspended Dopa.4U neurons showed IC50 values for tetrodotoxin of 27 and 2.9 nM, respectively. GABA-induced currents that could be observed in adherent Dopa.4U neurons could not be detected in suspended cells. Application of current pulses induced action potentials in approx. 70 % of the cells. Our results proved the feasibility of automated electrophysiological characterization of neuronal cells.

Neuronal in vitro activity is more sensitive to valproate than intracellular ATP: Considerations on conversion problems of IC50 in vitro data for animal replacement.

We investigated the effects of acute valproate (VPA) on mouse embryonic primary cortex cells (MEPCs). Intracellular ATP concentrations were compared with changes in the mean action potential (AP) frequencies of MEPC networks growing on microelectrode arrays. Our data implies biphasic reactions towards increasing VPA concentrations for both parameters. Intracellular ATP and mean AP frequencies increased around characteristic concentrations of 0.15 and 0.07mM to hormetic plateaus of approx. 120% and 160% of their controls, before fading around 17 and 1.7 mM, respectively. The biphasic in vitro behavior of the two parameters hinders a simple extraction of IC50 and Hillslope values. Different ways of data-fitting with single and double logistic functions are discussed. For a typical hormetic increase of 60% above control, IC50 and Hillslope were decreased by 37% and 15%, respectively. Despite these marginal effects at a logarithmic concentration scale, the hormetic and double logistic behavior of parameters may provide information on the mode of action of toxic compounds. Comparison of our values with the LD50 of mice, recalculated by normalization to body mass, suggests that a neurotoxic rather than a cytotoxic mechanism is killing the animals. The future use of cellular microsystems to replace animal experiments will motivate the development of new microsensors, as well as the consideration of newly accessible parameters in systems biology models.

Cell Monitoring and Manipulation Systems (CMMSs) based on Glass Cell-Culture Chips (GC³s).

We developed different types of glass cell-culture chips (GC³s) for culturing cells for microscopic observation in open media-containing troughs or in microfluidic structures. Platinum sensor and manipulation structures were used to monitor physiological parameters and to allocate and permeabilize cells. Electro-thermal micro pumps distributed chemical compounds in the microfluidic systems. The integrated temperature sensors showed a linear, Pt1000-like behavior. Cell adhesion and proliferation were monitored using interdigitated electrode structures (IDESs). The cell-doubling times of primary murine embryonic neuronal cells (PNCs) were determined based on the IDES capacitance-peak shifts. The electrical activity of PNC networks was detected using multi-electrode arrays (MEAs). During seeding, the cells were dielectrophoretically allocated to individual MEAs to improve network structures. MEA pads with diameters of 15, 20, 25, and 35 µm were tested. After 3 weeks, the magnitudes of the determined action potentials were highest for pads of 25 µm in diameter and did not differ when the inter-pad distances were 100 or 170 µm. Using 25-µm diameter circular oxygen electrodes, the signal currents in the cell-culture media were found to range from approximately -0.08 nA (0% O2) to -2.35 nA (21% O2). It was observed that 60-nm thick silicon nitride-sensor layers were stable potentiometric pH sensors under cell-culture conditions for periods of days. Their sensitivity between pH 5 and 9 was as high as 45 mV per pH step. We concluded that sensorized GC³s are potential animal replacement systems for purposes such as toxicity pre-screening. For example, the effect of mefloquine, a medication used to treat malaria, on the electrical activity of neuronal cells was determined in this study using a GC³ system.

Design and Characterization of a Sensorized Microfluidic Cell-Culture System with Electro-Thermal Micro-Pumps and Sensors for Cell Adhesion, Oxygen, and pH on a Glass Chip.

We combined a multi-sensor glass-chip with a microfluidic channel grid for the characterization of cellular behavior. The grid was imprinted in poly-dimethyl-siloxane. Mouse-embryonal/fetal calvaria fibroblasts (MC3T3-E1) were used as a model system. Thin-film platinum (Pt) sensors for respiration (amperometric oxygen electrode), acidification (potentiometric pH electrodes) and cell adhesion (interdigitated-electrodes structures, IDES) allowed us to monitor cell-physiological parameters as well as the cell-spreading behavior. Two on-chip electro-thermal micro-pumps (ETµPs) permitted the induction of medium flow in the system, e.g., for medium mixing and drug delivery. The glass-wafer technology ensured the microscopic observability of the on-chip cell culture. Connecting Pt structures were passivated by a 1.2 µm layer of silicon nitride (Si3N4). Thin Si3N4 layers (20 nm or 60 nm) were used as the sensitive material of the pH electrodes. These electrodes showed a linear behavior in the pH range from 4 to 9, with a sensitivity of up to 39 mV per pH step. The oxygen sensors were circular Pt electrodes with a sensor area of 78.5 µm(2). Their sensitivity was 100 pA per 1% oxygen increase in the range from 0% to 21% oxygen (air saturated). Two different IDES geometries with 30- and 50-µm finger spacings showed comparable sensitivities in detecting the proliferation rate of MC3T3 cells. These cells were cultured for 11 days in vitro to test the biocompatibility, microfluidics and electric sensors of our system under standard laboratory conditions.