Aligned electrospun fibers for neural patterning

OBJECTIVES: To test a 3D approach for neural network formation, alignment, and patterning that is reproducible and sufficiently stable to allow for easy manipulation. RESULTS: A novel cell culture system was designed by engineering a method for the directional growth of neurons. This uses NG108-15 neuroblastoma x glioma hybrid cells cultured on suspended and aligned electrospun fibers. These fiber networks improved cellular directionality, with alignment angle standard deviations significantly lower on fibers than on regular culture surfaces. Morphological studies found nuclear aspect ratios and cell projection lengths to be unchanged, indicating that cells maintained neural morphology while growing on fibers and forming a 3D network. Furthermore, fibronectin-coated fibers enhanced neurite extensions for all investigated time points. Differentiated neurons exhibited significant increases in average neurite lengths 96 h post plating, and formed neurite extensions parallel to suspended fibers, as visualized through scanning electron microscopy. CONCLUSIONS: The developed model has the potential to serve as the basis for advanced 3D studies, providing an original approach to neural network patterning and setting the groundwork for further investigations into functionality

Modification of a neuronal network direction using stepwise photo-thermal etching of an agarose architecture

Control over spatial distribution of individual neurons and the pattern of neural network provides an important tool for studying information processing pathways during neural network formation. Moreover, the knowledge of the direction of synaptic connections between cells in each neural network can provide detailed information on the relationship between the forward and feedback signaling. We have developed a method for topographical control of the direction of synaptic connections within a living neuronal network using a new type of individual-cell-based on-chip cell-cultivation system with an agarose microchamber array (AMCA). The advantages of this system include the possibility to control positions and number of cultured cells as well as flexible control of the direction of elongation of axons through stepwise melting of narrow grooves. Such micrometer-order microchannels are obtained by photo-thermal etching of agarose where a portion of the gel is melted with a 1064-nm infrared laser beam. Using this system, we created neural network from individual Rat hippocampal cells. We were able to control elongation of individual axons during cultivation (from cells contained within the AMCA) by non-destructive stepwise photo-thermal etching. We have demonstrated the potential of our on-chip AMCA cell cultivation system for the controlled development of individual cell-based neural networks

Substrate Micropatterning as a New in Vitro Cell Culture System to Study Myelination

Artículo de publicación ISIMyelination is a highly regulated developmental process whereby oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system ensheathe axons with a multilayered concentric membrane. Axonal myelination increases the velocity of nerve impulse propagation. In this work, we present a novel in vitro system for coculturing primary dorsal root ganglia neurons along with myelinating cells on a highly restrictive and micropatterned substrate. In this new coculture system, neurons survive for several weeks, extending long axons on defined Matrigel tracks. On these axons, myelinating cells can achieve robust myelination, as demonstrated by the distribution of compact myelin and nodal markers. Under these conditions, neurites and associated myelinating cells are easily accessible for studies on the mechanisms of myelin formation and on the effects of axonal damage on the myelin sheath.Regenerative Medicine and Nanomedicine Initiative of the Canadian Institutes of Health Research (CIHR) RMF-7028 FONDECYT 1080252 CIHR Ministry of Industry of Canada Rio Tinto Alcan Molson Foundatio

A novel polymeric microelectrode array for highly parallel, long-term neuronal culture and stimulation

Thesis (M. Eng.)--Harvard-MIT Division of Health Sciences and Technology, 2008.Includes bibliographical references (leaves 51-56).Cell-based high-throughput screening is emerging as a disruptive technology in drug discovery; however, massively parallel electrical assaying of neurons and cardiomyocites has until now been prohibitively expensive. To address this limitation, we developed a scalable, all-organic 3D microelectrode array technology. The cheap, disposable arrays would be integrated into a fixed stimulation and imaging setup, potentially amenable to automated handling and data analysis. A combination of activity-dependent plasticity, made possible by independent control of up to 64 stimulating electrodes, and, eventually, of substrate chemical patterning would be employed to constrain the neuronal culture network connectivity. In order to ensure longterm survival of the cultures, a bottom feeder layer of glial cells would be grown. In addition to high-throughput screening application, the polymeric microelectrode arrays and integrated stimulation systems were designed to allow the long-term study of synaptic plasticity, combining excellent long-term culture capabilities with a unique ability to independently control each electrode stimulation pattern. The resulting activity could be monitored optically, e,g, with calcium or voltage sensitive dyes, and the images could be stored and processed (possibly even in real time) within the same environment (LabView) as the stimulator. To fabricate the polymeric microelectrode array, we prepare a multilayered mask substrate, by reversibly bonding together two sheets of implant-grade polydimethylsiloxane (PDMS) sheets, with or without a glass coverslip between them. Thanks to PDMS self-adhesive properties the various layers are held together stably but reversibly. The mask is then laser-patterned, using either a standard CO2 laser or a 193 nm excimer laser.(cont.) The mask can then be adhered onto a glassy carbon or ITO electrode, and polypyrrole, doped with either hyaluronic acid or sodium dodecylbenzesulfonic acid, can be electrodeposited through it. Finally, the construct is removed from the deposition bath and the upper, sacrificial mask layer carefully peeled away. This fabrication method allows exquisite control overall 3D electrode geometry, is suitable to produce structures between one and several hundred micrometers in diameter, either filled or tubular, and scales extremely well, so that, for example, 384 by 64 electrodes arrays can be patterned in just a few minutes and grown in the same time as a single array.by Giovanni Talei Franzesi.M.Eng

Acidic Laminin: Molecular Mechanisms and Potential for Nervous Tissue Repair

Biomaterials have shown promise for treatment of injuries to the nervous system. Laminin, a glycoprotein, forms distinct polymers under neutral (pH 7; neutral laminin, nLam) or acidic (pH 4; acidic laminin, aLam) conditions (1, 2). aLam promotes significant axonal growth (2), making it of interest as a therapeutic for nervous tissue injuries. In this thesis, instead of as a substrate, we evaluate unbound aLam. In Chapter 2.1, we use an in vitro model system to investigate the mechanisms underlying aLam growth promotion. Results indicate: 1) laminin can act as a signaling molecule promoting outgrowth of adult neurons in vitro; 2) aLam is a more efficient promoter of outgrowth than nLam; 3) both polymers signal through α1 and α3 integrins without increasing their expression; 4) aLam, increases α3 integrins when α1 integrins are blocked; 6) aLam increases vinculin, a focal adhesion complex protein. These findings indicate that aLam promotes outgrowth by increasing integrin activation to enhance neurite outgrowth. In Chapter 2.2 microcontact printing and live imaging were combined to evaluate aLam’s effects on growth dynamics. Our results suggest: 1) neurons will adhere to stamps and grow in a directional manner in culture; 2) cells did not adhere or grow well during live imaging. Results indicate potential for directing neuronal outgrowth, but optimization is necessary to assess growth dynamics. Peripheral nerve injury (PNI) and spinal cord injury (SCI) are devastating. In Chapter 3.1 we investigate aLam’s treatment potential in PNI. Results indicate that aLam treatment: 1) increased presence of larger diameter axons; 2) facilitated compliance in treadmill walking; 3) alleviated autophagia; 4) did not affect motor function, axon number or myelination. These data show that aLam treatment elicits an axon growth response without affecting motor function recovery. Further research is needed to optimize treatment for functional improvements. Chapter 3.2 evaluates aLam treatment after SCI. Results show that aLam treatment: 1) did not affect axon regeneration; 2) decreased astrocyte activation; 3) did not affect neuropathic pain or motor outcomes. The data indicate treatment did not lead to functional improvements. Further research is needed to investigate the potential of aLam for SCI repair

MEMS micro-contact printing engines

This thesis investigates micro-contact printing (µCP) engines using micro-electro-mechanical systems (MEMS). Such engines are self-contained and do not require further optical alignment and precision manipulation equipment. Hence they provide a low-cost and accessible method of multilevel surface patterning with sub-micron resolution. Applications include the field of biotechnology where the placement of biological ligands at well controlled locations on substrates is often required for biological assays, cell studies and manipulation, or for the fabrication of biosensors. A miniaturised silicon µCP engine is designed and fabricated using a wafer-scale MEMS fabrication process and single level and bi-level µCP are successfully demonstrated. The performance of the engine is fully characterised and two actuation modes, mechanical and electrostatic, are investigated. In addition, a novel method of integrating the stamp material into the MEMS process flow by spray coating is reported. A second µCP engine formed by wafer-scale replica moulding of a polymer is developed to further drive down cost and complexity. This system carries six complementary patterns and allows six-level µCP with a layer-to-layer accuracy of 10 µm over a 5 mm x 5 mm area without the use of external aligning equipment. This is the first such report of aligned multilevel µCP. Lastly, the integration of the replica moulded engine with a hydraulic drive for controlled actuation is investigated. This approach is promising and proof of concept has been provided for single-level patterning

Network Level Manipulation of Neuronal Populations via Microtechnology: Epilepsy on a Chip

Recent studies indicate that oscillations between functional states (ex: ictal, post-ictal) in epilepsy are due to fluctuations in neuronal network firing patterns. However, current epilepsy models are often limited to non-mechanistically identifying the most likely anti-epileptic drug candidates. Therefore, expanding research to the network level is a promising way to examine the mechanisms underlying mental pathologies and possibly assess better ways to treat them. Microtechnology, which allows for control of the local microenvironment, is a reliable way to study whole networks, but is rarely applied to neurological disease. The objective of this project is to combine microtechnology with standard neuroscience techniques in an effort to create a platform for high throughput testing of anti-epileptic drugs. To achieve this, we create “epileptic” neuronal networks in vitro, characterize network morphology and phenotypic connectivity, and evaluate network activity modulation due to genetic manipulations related to epilepsy. This project focuses on the gene SCN1a, which codes for the voltage gated sodium channel Nav1.1. Mutations in SCN1a are linked to Generalized Epilepsy with Febrile Seizures Plus. The central hypothesis is that mutations in SCN1a affect activity properties of individual neurons, thus impacting recurrent activity in small networks, and that examining these networks may provide insight into pathways involved in seizure propagation

Bioanalytical applications of microfluidic devices

The first part of the thesis describes a new patterning technique--microfluidic contact printing--that combines several of the desirable aspects of microcontact printing and microfluidic patterning and addresses some of their important limitations through the integration of a track-etched polycarbonate (PCTE) membrane. Using this technique, biomolecules (e.g., peptides, polysaccharides, and proteins) were printed in high fidelity on a receptor modified polyacrylamide hydrogel substrate. The patterns obtained can be controlled through modifications of channel design and secondary programming via selective membrane wetting. The protocols support the printing of multiple reagents without registration steps and fast recycle times. The second part describes a non-enzymatic, isothermal method to discriminate single nucleotide polymorphisms (SNPs). SNP discrimination using alkaline dehybridization has long been neglected because the pH range in which thermodynamic discrimination can be done is quite narrow. We found, however, that SNPs can be discriminated by the kinetic differences exhibited in the dehybridization of PM and MM DNA duplexes in an alkaline solution using fluorescence microscopy. We combined this method with multifunctional encoded hydrogel particle array (fabricated by stop-flow lithography) to achieve fast kinetics and high versatility. This approach may serve as an effective alternative to temperature-based method for analyzing unamplified genomic DNA in point-of-care diagnostic

Micro-technologies to constrain neuronal networks

Micro-technologies broadly encompass a range of technologies that deal with the developmentof tools on the order of a few microns. These tools have made steady inroads into traditionalbiology and have helped probe the functioning of cells on the order of tens of microns.The objective of this work was to use engineering techniques to ask specific questions inneuroscience.Using two different techniques, namely microcontact printing and microfluidics we suc-cessfully restricted the spread of networks of neurons to defined geometries. In the formercase, we chose to restrict networks to 'ring' shaped geometries, in order to study emergentreverberating properties in the resulting network. Ring shaped neuronal networks displayedreverberatory activity upon brief stimulation. This reverberatory activity was enhancedwhen network inhibition was abolished pharmacologically. Finally the effect of varying geometric parameters on this form of network activity was assessed. Here we found that smallchanges in the geometry did not have any significant effect on the reverberatory activity.In the second case, we restricted networks of neurons inside microfluidic devices. Thesemicrofluidic devices were capable of maintaining two populations of neurons in a fluidicallyisolated manner. The two populations communicated via microgrooves that allowed axons to reach across either population. We integrated an electrophysiological framework onto themicrofluidic device such that one of the two populations could be electrically stimulated. Weshow, using calcium imaging it was possible to stimulate neurons inside these devices.In conclusion, we have demonstrated the use of micro-technologies to constrain neuronalnetworks to specific geometries. We show here the emergence of reverberation in 'ring'shaped networks. Finally, we also created a novel microfluidic platform to culture neuronsfor extended periods of time