The interplay between anatomical connectivity and dynamics in neural networks plays a key role in the functional properties of the brain and in the associated connectivity changes induced by neural diseases. However, a detailed experimental investigation of this interplay at both cellular and population scales in the living brain is limited by accessibility. Alternatively, to investigate the basic operational principles with morphological, electrophysiological and computational methods, the activity emerging from large in vitro networks of primary neurons organized with imposed topologies can be studied. Here, we validated the use of a new bio-printing approach, which effectively maintains the topology of hippocampal cultures in vitro and investigated, by patch-clamp and MEA electrophysiology, the emerging functional properties of these grid-confined networks. In spite of differences in the organization of physical connectivity, our bio-patterned grid networks retained the key properties of synaptic transmission, short-term plasticity and overall network activity with respect to random networks. Interestingly, the imposed grid topology resulted in a reinforcement of functional connections along orthogonal directions, shorter connectivity links and a greatly increased spiking probability in response to focal stimulation. These results clearly demonstrate that reliable functional studies can nowadays be performed on large neuronal networks in the presence of sustained changes in the physical network connectivity.

This study reports on a microfluidic platform on which single multicellular spheroids from malignant pleural mesothelioma (MPM), an aggressive tumor with poor prognosis, can be loaded, trapped and tested for chemotherapeutic drug response. A new method to detect the spheroid viability cultured on the microfluidic chip as a function of the drug concentration is presented. This approach is based on the evaluation of the caspase activity in the supernatant sampled from the chip and tested using a microplate reader. This simple and time-saving method does only require a minimum amount of manipulations and was established for very low numbers of cells. This feature is particularly important in view of personalised medicine applications for which the number of cells obtained from the patients is low. MPM spheroids were continuously perfused for 48 hours with cisplatin, one of the standard chemotherapeutic drugs used to treat MPM. The 50% growth inhibitory concentration of cisplatin in perfused MPM spheroids was found to be twice as high as in spheroids cultured under static conditions. This chemoresistance increase might be due to the continuous support of nutrients and oxygen to the perfused spheroids.

From the observations, it is possible to demonstrate that Hyaff-11, a hyaluronan-based scaffold, has potential for MSC implantation and that may have application for the treatment of early OA in humans.

Regeneration of functional connectivity within a neural network after different degrees of lesion is of utmost clinical importance. To test pharmacological approaches aimed at recovering from a total or partial damage of neuronal connections within a circuit, it is necessary to develop a precise method for controlled ablation of neuronal processes. We combined a UV laser microdissector to ablate neural processes in vitro at single neuron and neural network level with infrared holographic optical tweezers to carry out force spectroscopy measurements. Simultaneous force spectroscopy, down to the sub-pico-Newton range, was performed during laser dissection to quantify the tension release in a partially ablated neurite. Therefore, we could control and measure the damage inflicted to an individual neuronal process. To characterize the effect of the inflicted injury on network level, changes in activity of neural subpopulations were monitored with subcellular resolution and overall network activity with high temporal resolution by concurrent calcium imaging and microelectrode array recording. Neuronal connections have been sequentially ablated and the correlated changes in network activity traced and mapped. With this unique combination of electrophysiological and optical tools, neural activity can be studied and quantified in response to controlled injury at the subcellular, cellular, and network level.

The development, connectivity, and structural plasticity of neuronal networks largely depend on the directional growth of axonal growth cones (GCs). The morphology and 3-D profile of axons and GCs of primary hippocampal neurons, grown onto glass surfaces coated with poly-D-lysine (PDL) and micropatterned with stripes of the adhesion molecule L1 by using the indirect microcontact printing, were investigated. Neurons were fixed at early stages (one to seven days) of in vitro development prior to synapse formation, and analyzed by fluorescence and atomic force microscopy. The latter technique allowed us to investigate the 3-D morphology of the GCs, and detect their morphological rearrangements during axon outgrowth and during contact with the underlying substrate. We found that axons decreased their height-to-width ratio over development in culture, and that this value became particularly low when the axon and the GC proceeded onto a surface containing attracting cues such as L1 with respect to GCs growing onto a nonspecific adhesion substrate such as PDL. Along with this shape change of the axons, GCs lying onto L1 tracks displayed a flattened shape, ideal for sensing and progression, whereas GCs onto areas of nonspecific adhesion displayed more prominent shapes and steeper edges.

The ability of neurons to extend projections and to form physical connections among them (i.e., "connect-ability") is altered in several neuropathologies. The quantification of these alterations is an important read-out to investigate pathogenic mechanisms and for research and development of neuropharmacological therapies, however current morphological analysis methods are very time-intensive. Here, we present and characterize a novel on-chip approach that we propose as a rapid assay. Our approach is based on the definition on a neuronal cell culture substrate of discrete patterns of adhesion protein spots (poly-d-lysine, 23 ± 5 μm in diameter) characterized by controlled inter-spot separations of increasing distance (from 40 μm to 100 μm), locally adsorbed in an adhesion-repulsive agarose layer. Under these conditions, the connect-ability of wild type primary neurons from rodents is shown to be strictly dependent on the inter-spot distance, and can be rapidly documented by simple optical read-outs. Moreover, we applied our approach to identify connect-ability defects in neurons from a mouse model of 22q11.2 deletion syndrome/DiGeorge syndrome, by comparative trials with wild type preparations. The presented results demonstrate the sensitivity and reliability of this novel on-chip-based connect-ability approach and validate the use of this method for the rapid assessment of neuronal connect-ability defects in neuropathologies.

This study is the first to show electrical activity of SGNs on MEAs. Our findings may help to improve stimulation by and to reduce energy consumption of CIs and thereby contribute to the development of fully implantable devices with better auditory resolution in the future.

Abstract. We previously established a line of immortalized normal human articular chondrocytes, lbpva55, expressing the E6 and E7 transforming genes of the human papilloma virus type 16. With this study we investigated the phenotypic modulation ability of this cell line, cultured in different conditions, with the aim of validating its use for studies on cartilage metabolism and physiology. To this end, we performed a quantitative analysis, using real-time PCR technology, of the expression of the main structural components of the cartilage matrix (collagens I, II and aggrecan), of two transcription factors regulating chondrocyte differentiation (Sox-9 and Egr-1) and of some enzymes involved in matrix turnover (cathepsin B, MMP-1 and MMP-13). Results showed that, under defined conditions, lbpva55 cells were able to re-express the chondrocyte phenotype that was lost in a conventional monolayer condition, as demonstrated by an up-regulation of collagen II, the main marker of hyaline cartilage and Sox-9, a master gene regulator of chondrocytic differ-entiation. The gene expression profile of our immortalized cells compared with that of normal articular chondrocytes showed that this line could be used as a valid in vitro model for a better understanding of cell molecular mechanisms relevant for the development of new therapeutic approaches in rheumatic diseases and for the cartilage engineering field.