Soft Micro-Channels For Cell Culturing And Migration Studies

Various techniques and methods have been studied and developed to aid nerve regeneration and repairing nerve injuries. Among all, nerve grafting is the gold standard for bridging the gap between the injured nerve stumps. Despite the advantages of this technique, there are also various drawbacks that have encouraged the exploration of alternative, less invasive methods for promoting nerve regeneration. In this thesis, we have fabricated soft micro-channels for cell culturing and migration studies which could act as an interface capable of long-term, reliable, and high-resolution stimulation device for nerve regeneration. Micro-channels fabrication is performed using a ombination of photolithography technique and physical vapor deposition (PVD) methods. Initially, the surfaces of the micro-channels are treated with oxygen plasma to convert the surface of PDMS from hydrophobic to hydrophilic and to further provide an optimal environment for cells to adhere and grow. Next, in vitro studies were performed on the fabricated micro-channels to demonstrate feasibility of the platform to promote adherence and growth of PC12 cells (cell line derived from a pheochromocytomas of the rat adrenal medulla)

Functional nano-bio interfaces for cell modulation

Interacting cellular systems with nano-interfaces has shown great promise in promoting differentiation, regeneration, and stimulation. Functionalized nanostructures can serve as topological cues to mimic the extracellular matrix network to support cellular growth. Nanostructures can also generate signals, such as thermal, electrical, and mechanical stimulus, to trigger cellular stimulation. At this stage, the main challenges of applying nanostructures with biological systems are: (1) how to mimic the hierarchical structure of the ECM network in a 3D format and (2) how to improve the efficiency of the nanostructures while decreasing its invasiveness. To enable functional neuron regeneration after injuries, we have developed a 2D nanoladder scaffold, composed of micron size fibers and nanoscale protrusions, to mimic the ECM in the spinal cord. We have demonstrated that directional guidance during neuronal regeneration is critical for functional reconnection. We further transferred the nanoladder pattern onto biocompatible silk films. We established a self-folding strategy to fabricate 3D silk rolls, which is an even closer system to mimic the ECM of the spinal cord. As demonstrated by in vitro and in vivo experiments, such a scaffold can serve as a grafting bridge to guide axonal regeneration to desired targets for functional reconnection after spinal cord injuries. Benefited from the robust self-folding techniques, silk rolls can also be used for heterogeneous cell culture, providing a potential therapeutic approach for multiple tissue regeneration directions, such as bones, muscles, and tendons. For achieving neurostimulation, we have developed photoacoustic nanotransducers (PANs), which generate ultrasound upon excitation of NIR II nanosecond laser light. By surface functionalize PAN to bind to neurons, we have achieved an optoacoustic neuron stimulation process with a high spatial and temporal resolution, proved by in-vitro and in-vivo experiments. Such an application can enable non-invasive, optogenetics free and MRI compatible neurostimulation, which provides a new direction of gene-transfection free neuromodulation. Collectively, in this thesis, we have developed two systems to promote functional regeneration after injuries and stimulate neurons in a minimally invasive manner. By integrating those two functions, a potential new generation of the bioengineered scaffold can be investigated to enable functional and programmable control during the regeneration process

Design of an animal model for testing alginate tissue repair scaffolds in spinal cord injury

Current treatments for spinal cord injury (SCI) are extremely limited due to the fact that the central nervous system lacks the intrinsic ability to regenerate, and constitutes a poor environment for regenerative axon growth. Nerve tissue engineering is an emerging field with the aim of repairing or creating new nerve tissues to promote functional recovery by using artificial tissue repair scaffolds. The design of a stable and consistent animal model of SCI is essential to study the effectiveness of scaffolds in promoting nervous system repair. In this study, a partial transection animal model was created with a three dimensional lesion at T8-T9 that disrupts axonal pathways unilaterally in the dorsal columns of the rat spinal cord. Alginate hydrogel scaffolds incorporating living Schwann cells were fabricated to evaluate the abilities of those scaffolds to foster axonal regeneration. The surgical technique was improved to provide better outcomes related to bleeding during surgery, weight control, neurological function and surgery duration. The survival rate of animals during the surgical procedure and post-surgery period was ultimately increased to 100%. Histology and immunohistochemistry results indicated that implanted alginate scaffolds may induce larger cavities and extenuate harmful inflammation responses, but that effect was ameliorated by inclusion of Schwann cells in the scaffold. However, neither plain alginate scaffolds nor scaffolds containing living Schwann cells were able to improve regeneration of identified axon tracts in the spinal dorsal columns. This research also employed a synchrotron based x-ray phase contrast imaging technique coupled with computed-tomography to visualize the low optical density structural features of scaffolds and spinal cord tissues in formaldehyde fixed specimens. The imaging results suggest that this is a promising method for analyzing the structure of tissue repair scaffolds within the spinal cord. This degree of structural characterization, potentially applicable to living tissue, is not afforded by other conventional image analysis techniques

Developing an In Vitro Assay for Detection and Characterization of Functional Connectivity within Transplantation Candidate Embryonic Stem Cell-Derived V2a Interneuron Networks

Facilitating plasticity after spinal cord injury tends to be the focus of most modern interventions for this condition. In particular, stem cell therapies attempt to both modulate and mimic some of the native plasticity after injury through multiple mechanisms. One such mechanism, the creation of new exogenous relay circuits bridging the injury, has been explored extensively, revealing serious impediments to its optimization and adoption for clinical settings. Our collaborator, the Sakiyama-Elbert group, has spent years addressing the first limitation, the variability of cellular graft composition, by perfecting protocols to generate embryonic stem cell (ESC)-derived populations of neurons with pre-determined genetic identity. Recently, they developed a protocol to develop highly-enriched populations of Chx10-expressing V2a interneurons (INs), a ventral interneuron population that has garnered recent interest due to its role in central pattern generating function and favorable phenotypic properties. This predominantly glutamatergic and long, ipsilaterally projecting population appears to be a prime candidate for transplantation therapies for SCI, especially for the creation of relay circuits that can potentially circumnavigate injuries. The research documented in this thesis attempts to begin to address the second limitation of stem cell transplantation therapy, our minimal understanding of intra-graft network connectivity after transplantation. Due to the limitations of current techniques for evaluating the connectivity of populations like ESC-derived V2a INs, the relationship between functional recovery and the functional properties of the novel circuits formed within the graft still eludes researchers. This thesis focuses on the development of an assay capable of rapidly detecting connectivity within ESC-derived candidate populations. By extending previous work in the stem cell field, we combine in vitro multi-electrode arrays (MEAs) with an extensively studied metric of functional connectivity, cross-correlation, to detect and characterize individual functional connections between ESC-derived neurons. We first validated this assay by culturing ESC-derived populations differentiated for increased expression of Chx10 on MEAs. We found that both dissociated and aggregated cultures formed functional busting networks with significant functional connectivity detected with the use of Between-Sample Analysis of Connectivity, a methodology originally developed for in vitro circadian networks. Aggregated networks, however, had much more consistent electrode coverage and individual neuron detection that dissociated networks. After this validation study, we characterized the functional connectivity within highly-enriched populations of ESC-V2a INs, comparing their connectivity to populations of ESC-MN/glia and mixed populations of ESC-V2a/MN/glia. We found that ESC-MN/glia aggregates formed active networks with a variety of activity and functional connectivity that was dependent on the transmission of glutamate. ESC-V2a INs could only survive out to the 4-week time point if they were grown in media conditioned with glial factors, but these cultures still lacked spontaneous extracellular activity. Mixed ESC-V2a/MN/glia populations formed the most active networks and had thousands of detectable connections which were also dependent on glutamate transmission. Application of glycine antagonist modulated network activity but the underlying cause is fairly inconclusive due to possible secondary effects. High growth factor concentrations in the growth media actually decreased network activity and detectable functional connections in the mixed populations. All of these findings in this proof of concept study collectively suggest that a mixture of ESC-V2a INs and ESC-MN/glia may be the most viable candidate for transplantation and sets the stage for future investigations into the manipulability of their connectivity with electrical stimulation, as well as scaled versions of this assay performed in combination with animal studies

New strategies for peripheral nerve regeneration

Nerve repair is still a major challenge in surgery, regenerative medicine and tissue engineering even if progress has been made over the last 30 years. Functional recovery after severe lesions to a nerve is often incomplete and rarely totally successful. In this thesis I present a multi-disciplinary approach to improve the regenerative potential of “nerve repair tubes” that aim to reconnect wounded nerves and refine or replace autologous nerve graft, the clinical current gold standard. The efficacy of such tubes has already been shown in the clinic especially for small gap injuries, but the outcomes are still limited, and ought to be improved by e.g. micro/nano-topography, growth factor delivery systems, supportive cells or active features such as electrical stimulation, which have individually been shown to enhance nerve regeneration. In this study organotypic cultures of dorsal root ganglions (DRG) isolated from neonatal rats were used throughout as an in vitro model of nerve regeneration. Here I tested different devices in combination with growth factors to contribute to the fundamental and technical knowledge necessary to improve the regenerative potential of such tubes. I investigated the interaction between surface features and growth factors in their joined influence on regenerating DRGs. For this polydimethylsiloxane (PDMS), a polymer with adjustable elasticity was used together with photolithography to build devices of different stiffness with different surface microgrooves, on which DRG could be grown. To optimise the use of nerve growth factor (NGF) in conjunction with these devices, and to show how NGF interacts with stiffness and topography the reaction of the DRG was tested. To ease the making of three-dimensional internally microstructured tubes I have developed up a novel, timesaving, fabrication technique for polycaprolactone (PCL) “Swiss roll” nerve repair tubes. This technique improves the reproducibility of the scaffold, and using DRG its potential for nerve regeneration is being demonstrated. The influence of time-variant, balanced, pulsed electric stimulation is a potentially useful means to influence nerve regeneration. To narrow down the parameter space the effect of various electric fields was tested in their effect on DRG regeneration using commercially available devices. In collaboration with Christopher Martin from the School of Engineering, novel custom-made devices that allowed us to quantify the directional response of the regenerating axons were developed, and the guidance effect of pulsed alternating current (AC) electrical fields on regenerating DRGs axons was investigated in vitro. This approach allows in principle to transfer the use of this nerve guiding strategy to potentially improve nerve repair tubes

Microfluidic production of stem-cell microcapsules for spinal cord injury repair

Stem cell therapy demonstrates much promise for the replacement of damaged tissue in several diseases, including spinal cord injury. However, challenges around the control of stem cell fate in situ still hinders effective recovery of the normal tissue function. Stem cell encapsulation permits their immobilization within biocompatible scaffolds, allowing for a better control of parameters such as proliferation, integration, migration and differentiation within the host tissue. A customized microfluidic device was developed for the production of alginate microcapsules. The diameter of such microcapsules could be easily controlled by the modification of the fluids flow rates, allowing for the reproducible production of highly monodisperse microcapsules. This microfluidic method was then successfully applied for the encapsulation of two different types of stem cells: (i) Neural Stem Cells and (ii) Dental Pulp Stem Cells. Both cell types demonstrated survival within the alginate microcapsules for up to three weeks in culture. However, an early egress of cells from inside to outside of the microcapsules was observed 3 days post-encapsulation. In order to delay such cell escape, alginate microcapsules were modified through the addition of type I collagen. The alginate-collagen microcapsules permitted similar rates of cell survival and permitted the delay of cell egress until 10 days after encapsulation. Stem cells demonstrated a retention of their stem cell and neuronal differentiation properties upon selective release from alginate-collagen microcapsules, as demonstrated by high proliferation rates and the production of stem cell and neuronal markers. When cell-laden microcapsules were transplanted into an ex vivo SCI model the microcapsules were able to effectively retain the transplanted stem cells at the site of implantation. Transplanted cells survived up to 10 days in culture after transplantation and demonstrated the production of neuronal markers within the cord cultures. The results presented in this thesis demonstrate the ability of stem cells to retain their viability and neuronal differentiation capacity within alginate-collagen microcapsules, thereby providing a promising future therapy for the treatment of spinal cord injury

Electromyogram Interference Reduction In Neural Signal Recording Using Simple RC Compensation Circuits

Neuroprosthesis can partially restore lost motor functionalities of individuals such as bladder voiding using functional electrical stimulation (FES) techniques. FES involves applying pattern of electrical current pulses using implanted electrodes to trigger affected nerves that are damaged due to paralysis. A neural signal recorded using tripolar cuff electrodes is significantly contaminated due to the presence of EMG interference from the surrounding muscles. Conventional neural amplifiers are unable to remove such interferences and modifications to the design are required. The modification to the design of the Quasi-tripole (QT) amplifier is considered in this work to minimise the EMG interferences from neural signal recording. The analogy between this modified version of QT known as mQT and Wheatstone bridge claims to neutralise the EMG interference by adding compensation circuit to either end of the outer electrodes of the tripolar cuff and therefore balancing the bridge. In this work, we present simple 3 and 2 stage RC compensation circuits to minimise EMG interference in trying to balance the bridge in the neural frequency band of interest (500-10kHz). It is shown that simple RC compensation circuit in series reduces EMG interference only at the spot frequency rather than linearly in the entire frequency band of interest. However, two and three stages RC ladder compensation circuits mimicking electrode-electrolyte interface, can minimize the EMG interference linearly in the entire frequency band of interest, without requiring any readjustment to their components. The aim is to minimise EMG interference as close to null as possible. Invitro testing of about 20% imbalanced cuff electrode with proposed 3 and 2 stage RC ladder compensation circuits resulted in linear EMG interference reduction atleast by a factor of 6. On an average, this yielded an improvement of above 80% EMG minimisation, in contrast to above 90% observed in the optimisation results, when 1Ω transimpedance (EMG) was introduced into the setup. Further improvements to the setup and design can give more promising results in reliable neural signal recording for FES applications

Engineered environments for biomedical applications: anisotropic nanotopographies and microfluidic devices

During the last two decades micro- and nano-fabrication techniques originally developed for electronic engineering have directed their attention towards life sciences. The increase of analytical power of diagnostic devices and the creation of more biomimetic scaffolds have been strongly desired by these fields, in order to have a better insight into the complexity of physiological systems, while improving the ability to model them in vitro. Technological innovations worked to fill such a gap, but the integration of these fields of science is not progressing fast enough to satisfy the expectations. In this thesis I present novel devices which exploit the unique features of the micro- and nanoscale and, at the same time, match the requirements for successful application in biomedical research. Such biochips were used for optical detection of water-dispersed nanoparticles in microchannels, for highly controlled cell-patterning in closed microreactors, and for topography-mediated regulation of cell morphology and migration. Moreover, pilot experiments on the pre-clinical translation of micropatterned scaffolds in a rat model of peripheral nerve transaction were initiated and are ongoing. Given these results, the devices presented here have the potential to achieve clinical translation in a short/medium time, contributing to the improvement of biomedical technologies

Real-time bioimpedance measurements of stem cellbased disease models-on-a-chip

In vitro disease models are powerful platforms for the development of drugs and novel therapies. Stem-cell based approaches have emerged as cutting-edge tools in disease modelling, allowing for deeper insights into previously unknown disease mechanisms. Hence the significant role of these disease-in-a-dish methods in therapeutics and translational medicine. Impedance sensing is a non-invasive, quantitative technique that can monitor changes in cellular behaviour and morphology in real-time. Bioimpedance measurements can be used to characterize and evaluate the establishment of a valid disease model, without the need for invasive end-point biochemical assays. In this work, two stem cell-based disease models-on-a-chip are proposed for acute liver failure (ALF) and age-related macular degeneration (AMD). The ALF disease model-on-a-chip integrates impedance sensing with the highly-differentiated HepaRG cell line to monitor in real-time quantitative and dynamic response to various hepatotoxins. Bioimpedance analysis and modelling has revealed an unknown mechanism of paracetamol hepatotoxicity; a temporal, dose-dependent disruption of tight junctions (TJs) and cell-substrate adhesion. This disruption has been validated using ultrastructural imaging and immunostaining of the TJ-associated protein ZO-1. Age-related macular degeneration (AMD) is the leading cause of blindness in the developed world with a need for disease models for its currently incurable forms. Human induced pluripotent stem cells (hiPSCs) technology offers a novel approach for disease modelling, with the potential to impact translational retinal research and therapy. Recent developments enable the generation of Retinal Pigment Epithelial cells from patients (hiPSC-RPE), thus allowing for human retinal disease in vitro studies with great clinical and physiological relevance. In the current study, the development of a tissue-on- a-chip AMD disease model has been established using RPE generated from a patient with an inherited macular degeneration (case cell line) and from a healthy sibling (control cell line). A reproducible Electric Cell-substrate Impedance Sensing (ECIS) electrical wounding assay was conducted to mimic RPE damage in AMD. First, a robust and reproducible real-time quantitative monitoring over a 25-day period demonstrated the establishment and maturation of RPE layers on microelectrodes. A spatially-controlled RPE layer damage that mimicked cell loss in AMD was then initiated. Post recovery, significant differences in migration rates were found between case and control cell lines. Data analysis and modelling suggested this was due to the lower cell-substrate adhesion of the control cell line. These findings were confirmed using cell adhesion biochemical assays. Moreover, different-sized, individually-addressed square microelectrode arrays with high spatial resolution were designed and fabricated in-house. ECIS wounding assays were performed on these chips to study immortalized RPE migration. Migration rates comparable to those obtained with ECIS circular microelectrodes were determined. The two proposed disease-models-on-a-chip were then used to explore the therapeutic potential of the antioxidant N-Acetyl-Cysteine (NAC) on hiPSC-RPE and HepaRG cell recovery. Addition of 10 mM NAC at the end of a 24h paracetamol challenge caused a slight increase in the measured impedance, suggesting partial cell recovery. On the other hand, no effect on case hiPSC-RPE migration has been observed. More experiments are needed to examine the effect of different NAC concentrations and incubation periods. The therapeutic potential of electrical stimulation has also been explored. A preliminary study to evaluate the effect of electrical stimulation on RPE migration has been conducted. An externally applied direct current electric field (DC EF) of 300 mV/mm was found to direct the migration of the immortalized RPE cell line (hTERT-RPE1) perpendicular to the EF. The cells were also observed to elongate and to realign their long axes perpendicular to the applied EF. The proposed tissue-on-a-chip disease models are powerful platforms for translational studies. The potential of such platforms has been demonstrated through revealing unknown effects of acetaminophen on the liver as well as providing deeper insights into the underlying mechanisms of macular degeneration. Combining stem cell technology with impedance sensing provides a high throughput platform for studying patient-specific diseases and evaluating potential therapies