Endothelial and epithelial cellular barriers play a vital role in the selective transport of solutes and other molecules. The properties and function of these barriers are often affected in case of inflammation and disease. Modelling cellular barriers in vitro can greatly facilitate studies of inflammation, disease mechanisms and progression, and in addition, can be exploited for drug screening and discovery. Here, we report on a parallelizable microfluidic platform in a multiwell plate format with ten independent cell culture chambers to support the modelling of cellular barriers co-cultured with 3D tumor spheroids. The microfluidic platform was fabricated by microinjection molding. Electrodes integrated into the chip in combination with a FT-impedance measurement system enabled transepithelial/transendothelial electrical resistance (TEER) measurements to rapidly assess real-time barrier tightness. The fluidic layout supports the tubeless and parallelized operation of up to ten distinct cultures under continuous unidirectional flow/perfusion. The capabilities of the system were demonstrated with a co-culture of 3D tumor spheroids and cellular barriers showing the growth and interaction of HT29 spheroids with a cellular barrier of MDCK cells.

HepaChip microplate (HepaChip-MP) is a microfluidic platform comprised of 24 independent culture chambers with continuous, unidirectional perfusion. In the HepaChip-MP, an automated dielectrophoresis process selectively assembles viable cells into elongated micro tissues. Freshly isolated primary human hepatocytes (PHH) and primary human liver endothelial cells (HuLEC) were successfully assembled as cocultures aiming to mimic the liver sinusoid. Minimal quantities of primary human cells are required to establish micro tissues in the HepaChip-MP. Metabolic function including induction of CYP enzymes in PHH was successfully measured demonstrating a high degree of metabolic activity of cells in HepaChip-MP cultures and sufficient sensitivity of LC-MS analysis even for the relatively small number of cells per chamber. Further, parallelization realized in HepaChip-MP enabled the acquisition of dose-response toxicity data of diclofenac with a single device. Several unique technical features should enable a widespread application of this in vitro model. We have demonstrated fully automated preparation of cell cultures in HepaChip-MP using a pipetting robot. The tubeless unidirectional perfusion system based on gravity-driven flow can be operated within a standard incubator system. Overall, the system readily integrates in workflows common in cell culture labs. Further research will be directed towards optimization of media composition to further extend culture lifetime and study oxygen gradients and their effect on zonation within the sinusoid-like microorgans. In summary, we have established a novel parallelized and scalable microfluidic in vitro liver model showing hepatocyte function and anticipate future in-depth studies of liver biology and applications in pre-clinical drug development.

Real-time monitoring of the cell biomaterial interface for biomedical applications can significantly improve the assessment of biomaterials. However, currently there are no multiparametric real-time monitoring systems for the ex vivo assessment of biomaterials. This study presents a method for monitoring of cell and tissue response to biomaterials based on microsensor read-outs of pH, O-2, and impedance. In vitro, the sensors are compatible with cells implants. For ex vivo measurements, either a biodegradable polymer poly(lactic-co-glycolic acid) or a protein-repellent plasma polymer nanofilm is coated onto sensor arrays and placed on the chorioallantoic membrane of avian embryo ex ovo cultures. Signals related to dissolved oxygen, pH, and electrical impedance are continuously acquired for up to 90 h. Optical observation, immunohistochemical examination of CD45, and Haematoxylin and Eosin (H&E) staining show different unspecific immune responses. Tissue inflammation, capsule formation, and neovascularization are observed. These results correlate well with the type of coating material and the signal patterns acquired by the chemosensors. In addition, tissue death can be inferred from sensor signal patterns. This system is intended to enable continuous monitoring of biomaterials and is envisioned as a prescreening tool to provide real-time monitoring of cell/biomaterial interface and also to reduce animal testing for biomaterial assessment purposes.

Event Abstract Back to Event In Vitro And In Vivo Probes With Mushroom-shaped Microelectrodes - Tools For In-cell Electrophysiology Peter D. Jones1, Clemens Barthold2, Meike Beer1, Claus Burkhardt2, Katja Gutöhrlein3, Gerhard Heusel2, Udo Kraushaar4, Pranoti Kshirsagar2, Manuel Martina2, Sebastian Roehler2, Ramona Samba1, Birgit Schroeppel2, Angelika Stumpf2, Simon Werner1 and Martin Stelzle1* 1 Natural and Medical Sciences Institute at the University Tübingen, BioMEMS and Sensors, Germany 2 Natural and Medical Sciences Institute at the University Tübingen, Microsystems / Nanotechnology, Germany 3 Natural and Medical Sciences Institute at the University Tübingen, Microsystems / Nanotechnology, Germany 4 Natural and Medical Sciences Institute at the University Tübingen, Electrophysiology, Germany Motivation An ambitious goal for neuroscience and cardiac research is to apply microelectrode array (MEA) technology to simultaneously record from large cell populations with signal quality matching intracellular patch clamp recordings (Spira & Hai, 2013). Conventional MEAs record extracellular field action potentials, but cannot measure details such as subthreshold synaptic potentials. MEA-based cardiac electrophysiology struggles to resolve details of cardiac action potentials. Patch clamp techniques can resolve such details, but cannot record and stimulate for long times, and even skilled operators achieve low throughput with neural and cardiac cells. Inspired by the shape of dendritic spines, MEAs with mushroom-shaped microelectrodes have enabled 'in-cell' recordings of monophasic action potentials and subthreshold activity, based on enhanced coupling between cells and extracellular electrodes (Hai et al., 2009; Spira & Hai, 2013). Here, we present reliable methods to produce MEAs with micrometer-scale, three-dimensional, mushroom-shaped electrodes for in vitro and in vivo investigations towards high density in cell recordings. Materials and Methods Mushroom-shaped electrodes were integrated on in vitro and in vivo MEAs (Figure 1) by electroplating gold through micrometer-sized holes in the insulator and a sacrificial layer. In vitro MEAs on glass used silicon nitride (SiNx) as insulator and photoresist (AZ ECI 3027) as sacrificial layer. In vivo devices were fabricated on polyimide (PI) on glass handling substrates with PI as insulator and SiNx as sacrificial layer. Holes for mushrooms were produced by photolithography and plasma etching. Electrical leads were gold with titanium for adhesion on both sides. Electroplating was controlled to yield mushroom cap diameters below 2.5 µm. After electroplating, sacrificial layers were removed: photoresist by KOH solution and SiNx by CF4 plasma. Gold mushrooms were optionally coated with poly(3,4-ethylenedioxythiophene) (PEDOT) by electropolymerization (Gerwig et al., 2012). Before cell plating, in vitro MEAs were coated with polyethylene imine and either laminin or fibronectin. Cardiomyocytes derived from human induced pluripotent stem (hiPS) cells (Cellartis ChiPSC22) were cultivated for 6 days before recordings. Recordings were performed at 37 °C (MEA2100 amplifier, sampling 20 kHz, bandwidth 0.1–3000 Hz). Implantation of flexible in vivo probes was tested in agarose brain models similar to previously reported methods (Kozai and Kipke, 2009). Results Gold mushroom electrodes with cap diameters below 2.5 µm were reliably integrated on in vitro MEAs and 8 µm thick flexible in vivo probes (Figure 2). Gold mushroom surface areas of ~20 µm2 were confirmed by cyclic voltammetry in 0.5 M sulfuric acid (Trasatti & Petrii, 1992). These electrodes have an estimated impedance at 1 kHz (Z1 kHz) of 16 MΩ based on a specific capacitance of 0.5 pF/µm2 (Mirsky et al., 1997), although the system capacitance prevents measurement of this impedance. Conformal electrodeposition of PEDOT (Figure 2H) reduced impedance at 1 kHz to 700 kΩ while maintaining small electrode dimensions. Recordings from cardiomyocytes with gold mushroom electrodes (Figure 3) spontaneously showed monophasic action potentials with amplitudes of up to 1.3 mV, in contrast to similar recordings obtained only after electrical stimulation (Fendyur & Spira, 2012; Xie et al., 2012). We interpret these monophasic signals as indicating enhanced coupling between cells and electrodes. The recorded waveforms were attenuated from the intracellular potential, and further investigation is required to understand the mechanisms required to record monophasic signals; these points are discussed below. Flexible probes were implanted into agarose brain models by adhesive-free shuttles (Figure 4). Impedance measurements of PEDOT-coated mushrooms showed no damage due to implantation. Discussion Our processes reliably integrate mushroom electrodes with diameters below 2.5 µm on in vitro MEAs and flexible in vivo probes. Such electrodes should encourage engulfment by cells, thereby increasing seal resistance of cells on the electrodes; their shape and dimensions should enable suitable engulfment by mammalian neurons. For comparison, diameters of 3.5 µm would be unsuitable (Ojovan et al., 2015) and mushrooms are preferred over cylindrical electrodes (Santoro et al., 2014). A critical factor for in-cell recording is to reduce junctional membrane impedance. This has been achieved with Aplysia neurons by biochemical functionalization (Hai et al., 2010) or electroporation (Hai & Spira, 2012), and with rodent cardiomyocytes by electroporation on mushroom electrodes (Fendyur & Spira, 2012) or nanowire electrodes (Xie et al., 2012). Consistent with work pioneered by the lab of Micha Spira, we recorded large signals from cardiomyocytes despite using small, high impedance electrodes. Surprisingly, we observed monophasic action potentials without special consideration to reduce membrane impedance. No electroporation was needed to record monophasic signals, in contrast to other reports (Fendyur & Spira, 2012; Xie et al., 2012). The mechanisms of engulfment and reduction of junctional membrane impedance which lead to these results must be further investigated. The recorded monophasic action potentials (Figure 3) were attenuated and distorted versus the intracellular potential. Attenuation depends on electrical coupling and seal resistance (Ojovan et al., 2015). Additionally, capacitance of insulated conducting leads was 10–50 pF, which is large when compared to the gold mushroom electrodes (estimated Z1 kHz = 16 MΩ, capacitance of 8 pF). The insulation may act as a low pass filter, shunting high frequency signals to the electrolyte to produce the observed waveform. PEDOT coatings should minimize this problem but the interaction between cells and PEDOT mushrooms has not yet been investigated. Investigation of mushroom electrodes on in vivo probes is still required. Engulfment could help to achieve stable single unit recordings. The use of flexible substrates will be critical to minimize motion between electrodes and cells. Micromotions of tens of micrometers, many times larger than our electrodes themselves, have been measured in rat cortex (Gilletti & Muthuswamy, 2006). PEDOT coatings with biochemical functionalization can encourage recognition by specific cells (Zhu et al., 2014). Similar functionalization could encourage interactions such as engulfment by specific cell types and recruitment of ion channels to reduce membrane impedance. Conclusion We have developed reliable methods to produce both in vitro and in vivo MEAs with integrated mushroom-shaped microelectrodes as tools to study in-cell recording of electrical activity. Recordings of monophasic action potentials from cardiomyocytes support the possibility of MEA-based in-cell recording. These tools can be applied to reveal and improve the mechanisms by which cells recognize and interact with 3D mushroom microelectrodes, moving us closer to the goal of stable, patch-clamp-quality signals from large cell populations with MEA technology. Acknowledgements This work was funded by the European Commission (FP7 Information and Communication Technologies, Future Emerging Technology programme, BRAINLEAP grant n. 306502). Helpful discussions with Stefano Ferraina, Luc Gentet, Michele Giugliano, and Micha Spira are acknowledged. We thank Sandra Buckenmaier for preparing cell cultures. References Fendyur, A., and Spira, M. E. (2012). Toward on-chip, in-cell recordings from cultured cardiomyocytes by arrays of gold mushroom-shaped microelectrodes. Front. Neuroeng. 5, 21. doi:10.3389/fneng.2012.00021. Gerwig, R., Fuchsberger, K., Schroeppel, B., Link, G. S., Heusel, G., Kraushaar, U., et al. (2012). PEDOT-CNT Composite Microelectrodes for Recording and Electrostimulation Applications: Fabrication, Morphology, and Electrical Properties. Front. Neuroeng. 5, 8. doi:10.3389/fneng.2012.00008. Gilletti, A., and Muthuswamy, J. (2006). Brain micromotion around implants in the rodent somatosensory cortex. J. Neural Eng. 3, 189–95. doi:10.1088/1741-2560/3/3/001. Hai, A., Dormann, A., Shappir, J., Yitzchaik, S., Bartic, C., Borghs, G., et al. (2009). Spine-shaped gold protrusions improve the adherence and electrical coupling of neurons with the surface of micro-electronic devices. J. R. Soc. Interface 6, 1153–65. doi:10.1098/rsif.2009.0087. Hai, A., Shappir, J., and Spira, M. E. (2010). Long-term, multisite, parallel, in-cell recording and stimulation by an array of extracellular microelectrodes. J. Neurophysiol. 104, 559–68. doi:10.1152/jn.00265.2010. Hai, A., and Spira, M. E. (2012). On-chip electroporation, membrane repair dynamics and transient in-cell recordings by arrays of gold mushroom-shaped microelectrodes. Lab Chip 12, 2865. doi:10.1039/c2lc40091j. Kozai, T. D. Y., and Kipke, D. R. (2009). Insertion shuttle with carboxyl terminated self-assembled monolayer coatings for implanting flexible polymer neural probes in the brain. J. Neurosci. Methods 184, 199–205. doi:10.1016/j.jneumeth.2009.08.002. Mirsky, V. M., Riepl, M., and Wolfbeis, O. S. (1997). Capacitive monitoring of protein immobilization and antigen-antibody reactions on monomolecular alkylthiol films on gold electrodes. Biosens. Bioelectron. 12, 977–989. doi:10.1016/S0956-5663(97)00053-5. Ojovan, S. M., Rabieh, N., Shmoel, N., Erez, H., Maydan, E., Cohen, A., et al. (2015). A feasibility study of multi-site, intracellular recordings from mammalian neurons by extracellular gold mushroom-shaped microelectrodes. Sci. Rep. 5, 14100. doi:10.1038/srep14100. Santoro, F., Dasgupta, S., Schnitker, J., Auth, T., Neumann, E., Panaitov, G., et al. (2014). Interfacing Electrogenic Cells with 3D Nanoelectrodes: Position, Shape, and Size Matter. ACS Nano 8, 6713–6723. doi:10.1021/nn500393p. Spira, M. E., and Hai, A. (2013). Multi-electrode array technologies for neuroscience and cardiology. Nat. Nanotechnol. 8, 83–94. doi:10.1038/nnano.2012.265. Trasatti, S., and Petrii, O. A. (1992). Real surface area measurements in electrochemistry. J. Electroanal. Chem. 327, 353–376. doi:10.1016/0022-0728(92)80162-W. Xie, C., Lin, Z., Hanson, L., Cui, Y., and Cui, B. (2012). Intracellular recording of action potentials by nanopillar electroporation. Nat. Nanotechnol. 7, 185–190. doi:10.1038/nnano.2012.8. Zhu, B., Luo, S.-C., Zhao, H., Lin, H.-A., Sekine, J., Nakao, A., et al. (2014). Large enhancement in neurite outgrowth on a cell membrane-mimicking conducting polymer. Nat. Commun. 5, 4523. doi:10.1038/ncomms5523. Figure Legend Figure 1: Fabrication process for mushroom MEAs. Holes in the sacrificial layer and insulator were produced by photolithography and plasma etching to define the mushroom stalks. Electroplating through and beyond the holes produced mushroom-shaped gold electrodes, which were free-standing after removal of the sacrificial layer. In vitro MEAs were produced on glass substrates with silicon nitride insulators and photoresist as the sacrificial layer. In vivo MEAs were produced on polyimide substrates (supported by glass, not illustrated). The insulator was also polyimide, and the sacrificial layer was silicon nitride. Figure 2: Mushroom microelectrode arrays. A–D: Photos of MEAs and microscopic images of mushroom electrodes on (A, B) in vitro and (C, D) in vivo MEAs. The texture in (D) is the surface supporting the transparent device. The offset of the electrodes from the center was intentional. E–H: Scanning electron images of (E) an intact mushroom electrode and cross-sections of (F) an in vitro mushroom electrode, (G) an in vivo mushroom electrode, and (H) a mushroom electrode coated with PEDOT. Cross-sections were prepared by focused ion beam milling. Mushroom cap diameters were 2.0–2.2 µm (F–G). The gold mushroom in H had a diameter of 2.8 µm before conformal deposition of approximately 150 nm PEDOT. The scale bar in E applies to E–H. Figure 3: Cardiac action potentials. In comparison to intracellular action potentials recorded by patch clamp (A) and extracellular field potentials recorded by planar microelectrodes (B), the signal recorded with mushroom microelectrodes appears similar to a filtered and attenuated intracellular action potential. Similar signals were recorded on several electrodes; selected traces from the same experiment are shown in D. Figure 4: Implantation of flexible probes in agarose. Removable shuttles (A) were produced with hydrophilic self-assembled monolayer surfaces. Flexible probes were easily aligned on the shuttles (B) with a drop of ethanol. After evaporation, the adhesion with the shuttle was sufficient to lift the weight of the probe. The shuttles were used to implant probes into agarose gel (C). Wicking of water between the shuttle and probe allowed removal of the shuttle (D). Figure 1 Keywords: PEDOT, cardiomyocytes, microfabrication, in vivo probes, in vitro MEA, mushroom electrodes Conference: MEA Meeting 2016 | 10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016. Presentation Type: oral Topic: MEA Meeting 2016 Citation: Jones PD, Barthold C, Beer M, Burkhardt C, Gutöhrlein K, Heusel G, Kraushaar U, Kshirsagar P, Martina M, Roehler S, Samba R, Schroeppel B, Stumpf A, Werner S and Stelzle M (2016). In Vitro And In Vivo Probes With Mushroom-shaped Microelectrodes - Tools For In-cell Electrophysiology. Front. Neurosci. Conference Abstract: MEA Meeting 2016 | 10th International Meeting on Substrate-Integrated Electrode Arrays. doi: 10.3389/conf.fnins.2016.93.00076 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers' terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 22 Jun 2016; Published Online: 24 Jun 2016. * Correspondence: Dr. Martin Stelzle, Natural and Medical Sciences Institute at the University Tübingen, BioMEMS and Sensors, Reutlingen, Germany, martin.stelzle@nmi.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Peter D Jones Clemens Barthold Meike Beer Claus Burkhardt Katja Gutöhrlein Gerhard Heusel Udo Kraushaar Pranoti Kshirsagar Manuel Martina Sebastian Roehler Ramona Samba Birgit Schroeppel Angelika Stumpf Simon Werner Martin Stelzle Google Peter D Jones Clemens Barthold Meike Beer Claus Burkhardt Katja Gutöhrlein Gerhard Heusel Udo Kraushaar Pranoti Kshirsagar Manuel Martina Sebastian Roehler Ramona Samba Birgit Schroeppel Angelika Stumpf Simon Werner Martin Stelzle Google Scholar Peter D Jones Clemens Barthold Meike Beer Claus Burkhardt Katja Gutöhrlein Gerhard Heusel Udo Kraushaar Pranoti Kshirsagar Manuel Martina Sebastian Roehler Ramona Samba Birgit Schroeppel Angelika Stumpf Simon Werner Martin Stelzle PubMed Peter D Jones Clemens Barthold Meike Beer Claus Burkhardt Katja Gutöhrlein Gerhard Heusel Udo Kraushaar Pranoti Kshirsagar Manuel Martina Sebastian Roehler Ramona Samba Birgit Schroeppel Angelika Stumpf Simon Werner Martin Stelzle Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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Although electrosurgical instruments are widely used in surgery to cut tissue layers or to achieve hemostasis by coagulation (electrocautery), only little information is available concerning the inflammatory or immune response towards the debris generated. Given the elevated local temperatures required for successful electrocautery, the remaining debris is likely to contain a plethora of compounds entirely novel to the intracorporal setting. A very common in vitro method to study cell migration after mechanical damage is the scratch assay, however, there is no established model for thermomechanical damage to characterise cellular reactions. In this study, we established a new in vitro model to investigate exposure to high temperature in a carefully controlled cell culture system. Heatable thermostat-controlled aluminium stamps were developed to induce local damage in primary human umbilical vein endothelial cells (HUVEC). The thermomechanical damage invoked is reproducibly locally confined, therefore allowing studies, under the same experimental conditions, of cells affected to various degrees as well as of unaffected cells. We show that the unaffected cells surrounding the thermomechanical damage zone are able to migrate into the damaged area, resulting in a complete closure of the 'wound' within 48 h. Initial studies have shown that there are significant morphological and biological differences in endothelial cells after thermomechanical damage compared to the mechanical damage inflicted by using the unheated stamp as a control. Accordingly, after thermomechanical damage, cell death as well as cell protection programs were activated. Mononuclear cells adhered in the area adjacent to thermomechanical damage, but not to the zone of mechanical damage. Therefore, our model can help to understand the differences in wound healing during the early phase of regeneration after thermomechanical vs. mechanical damage. Furthermore, this model lends itself to study the response of other cells, thus broadening the range of thermal injuries that can be analysed.

In order to study possible toxic side effects of potential drug compounds in vitro a reliable test system is needed. Predicting liver toxicity presents a major challenge of particular importance as liver cells grown in a cell culture suffer from a rapid loss of their liver specific functions. Therefore we are developing a new microfluidic test system for liver toxicity. This test system is based on an organ-like liver 3D co-culture of hepatocytes and endothelial cells. We devised a microfluidic chip featuring cell culture chambers with integrated electrodes for the assembly of liver sinusoids by dielectrophoresis. Fluid channels enable an organ-like perfusion with culture media and test compounds. Different chamber designs were studied and optimized with regard to dielectrophoretic force distribution, hydrodynamic flow profile, and cell trapping rate using numeric simulations. Based on simulation results a microchip was injection-moulded from COP. This chip allowed the assembly of viable hepatocytes and endothelial cells in a sinusoid-like fashion.

Despite many research in the area of wireless sensor networks (WSNs) in recent years, the programming of sensor nodes is still time-consuming and tedious. One of the new paradigms which seem to be qualified to simplify the programming of sensor networks are service oriented architectures (SOAs). The composition of simple services to more complex ones can be a convenient way to design own applications. However, to enable the sophisticated techniques known from SOAs like replication and migration of services, a transactional model for WSNs is needed. In this paper, we compare the applicability of the standard commit protocol Two Phase Commit (2PC) and the Cross Layer Commit Protocol (CLCP) \cite{Obermeier2008a, Obermeier2008} to our scenario and present our own protocol Two Phase Commit with Caching (2PCwC). We show that the best results in terms of transmitted bytes per commit decision and memory consumption can be achieved if the appropriate protocol is chosen adaptively during runtime.

We have developed a microfluidic system - microPrep - for subcellular fractionation of cell homogenates based on dielectrophoretic sorting. Separation of mitochondria isolated from a human lymphoblastoid cell line was monitored by fluorescence microscopy and further characterized by western blot analysis. Robust high throughput and continuous long-term operation for up to 60 h of the microPrep chip system with complex biological samples became feasible as a result of a comprehensive set of technical measures: (i) coating of the inner surfaces of the chip with BSA, (ii) application of mechanical actuators to induce periodic flow patterns, (iii) efficient cooling of the device to ensure integrity of organelle, (iv) a wide channel to provide for high fluidic throughput, and (v) integration of a serial arrangement of 10 dielectrophoretic deflector units to enable separation of samples with a high particle load without clogging. Hence, microPrep yields tens of micrograms of enriched and purified mitochondria within hours. Western blots of mitochondria fractions showed that contaminating endoplasmatic reticulum was reduced by a factor 6 when compared with samples prepared by state of the art centrifugation.

Different approaches have been proposed on the use of digital and interactive technology in mobile learning contexts. In line with recent findings in the literature, we suggest that mobile learning systems benefit from an iterative design and development process within a holistic, socio-technical system view. This system view makes allowance for the complex dynamics between teachers, students, researchers, the multiplicity of contextual factors, and the specifications and requirements of the digital devices and applications, as well as their interrelations. Iterative design and development incorporates evaluation results of mobile learning scenarios that were designed according to system-based analyses. The school projects described in this paper, as well as their evaluation results, served as an integral part in creating a dynamic optimization process for the design and development of digital and interactive systems for mobile learning contexts in primary and secondary schools.

The relationship between mirror neuron systems and imitation is being widely studied. However, most if not all, studies on imitation have investigated only the mirror mode. The present study examined whether imitation in a mirror (specular) mode is likely to reflect similar or distinct neural processes and psychological principles as imitation in a non-mirror (anatomical) mode. Experiment 1 examined whether altering sensory information may reverse the typical mirror mode advantage, resulting in superior performance in the non-mirror mode. Experiment 2 examined whether the two different modes of imitation rely differentially on target selection (goals) and effector selection (means). Experiment 3 examined whether spatial translations are likely to occur in a typical non-mirror imitation mode. Experiment 4 examined whether non-mirror imitation would be the naturally selected mode of imitation under some situations. Findings from all experiments demonstrated marked differences between mirror and non-mirror modes of imitation. The implications of these findings may raise challenges for theories and models of mirror neurons.