Microfabricated Physiological Models for In Vitro Drug Screening Applications

Microfluidics and microfabrication have recently been established as promising tools for developing a new generation of in vitro cell culture microdevices. The reduced amounts of reagents employed within cell culture microdevices make them particularly appealing to drug screening processes. In addition, latest advancements in recreating physiologically relevant cell culture conditions within microfabricated devices encourage the idea of using such advanced biological models in improving the screening of drug candidates prior to in vivo testing. In this review, we discuss microfluidics-based models employed for chemical/drug screening and the strategies to mimic various physiological conditions: fine control of 3D extra-cellular matrix environment, physical and chemical cues provided to cells and organization of co-cultures. We also envision future directions for achieving multi-organ microfluidic devices

Recent developments in microfluidic technologies for central nervous system targeted studies

Neurodegenerative diseases (NDs) bear a lot of weight in public health. By studying the properties of the blood-brain barrier (BBB) and its fundamental interactions with the central nervous system (CNS), it is possible to improve the understanding of the pathological mechanisms behind these disorders and create new and better strategies to improve bioavailability and therapeutic efficiency, such as nanocarriers. Microfluidics is an intersectional field with many applications. Microfluidic systems can be an invaluable tool to accurately simulate the BBB microenvironment, as well as develop, in a reproducible manner, drug delivery systems with well-defined physicochemical characteristics. This review provides an overview of the most recent advances on microfluidic devices for CNS-targeted studies. Firstly, the importance of the BBB will be addressed, and different experimental BBB models will be briefly discussed. Subsequently, microfluidic-integrated BBB models (BBB/brain-on-a-chip) are introduced and the state of the art reviewed, with special emphasis on their use to study NDs. Additionally, the microfluidic preparation of nanocarriers and other compounds for CNS delivery has been covered. The last section focuses on current challenges and future perspectives of microfluidic experimentation.info:eu-repo/semantics/publishedVersio

Multifunctional nanostructures for intracellular delivery and sensing in electrogenic cells

In electrophysiology, multielectrode array devices (MEA) are the gold standard for the study of large ensambles of electrogenic cells. In the last decades, thanks to the adoption of nanotechnologies, the study of physiological and pathological conditions of electro-active cells in culture have becomes increasingly accurate. In parallel, studies exploited the integration of nanostructures with delivering capabilities with single-cell specificity and high throughput in biosensing platforms. Delivery and recording have independently led to great advances in neurobiology, however, their integration on a single chip would give complete insights into pathologies development and fundamental advancements in drug screening methods. In this work, we demonstrate how a microfluidic-MEA technology may be used to record both spontaneous and chemically induced activity in vitro. We propose a device that can deliver molecules to only a few chosen cells and detecting the response in cellular activity at multiple sites simultaneously. In addition, will be discussed how the adoption of nanoporous metamaterial in place of nanostructures might lower costs and speed up production. Furthermore, this same material, will be identified for the first time in this work as photoelectrical modulating material for eliciting electrogenic cells firing activity. Specifically, by converting NIR laser pulses into stimulatory currents, plasmonic metamaterials may be employed to induce action potentials. This method enables remote access to optical pacing with precise spatiotemporal control, allowing to be used as a valid alternative of the traditional genetic-based optical stimulation techniques. Therefore, in addition to pharmaceutical applications, these final characteristics may pave the way for a new generation of minimally invasive, cellular type-independent all-optical plasmonic pacemakers and muscle actuators

Multifunctional nanostructures for intracellular delivery and sensing in electrogenic cells

Biological studies on in vitro cell cultures are of fundamental importance for investigating cell response to external stimuli, such drugs for specific treatments, or for studying communication between cells. In the electrophysiology field, multielectrode array devices (MEA) are the gold standard for the study of large ensambles of electrogenic cells. Thus, their improvement is a central topic nowadays in neuroscience and cardiology [1]. In the last decades, thanks to the adoption of nanotechnologies, the study of physiological and pathological conditions of electro-active cells in culture have becomes increasingly accurate[2], allowing for monitoring action potentials from many cells simultaneously. In fact, nanoscale biomaterials were able to overcome the limitations of previous technologies, paving the way to the development of platforms for interfacing the electrogenic cells at unprecedented spatiotemporal scales. These devices, together with microfluidics, are starting to be used for drug screening and pharmaceutical drug development since they represent a powerful tool for monitoring cell response when cultures are stimulated by target compounds. Many pharmaceutical agents, however, including various large molecules (enzymes, proteins, antibodies) and even drug-loaded pharmaceutical nanocarriers, need to be delivered intracellularly to exercise their therapeutic action inside the cytoplasm[3]. Nanoscale electrodes offer individual cell access and non-destructive poration of the cellular membrane enabling high capability in the delivery of biomolecules. Among all the techniques, electroporation have proven encouraging potential as alternative to the carrier mediated methods for molecular delivery into cultured cells[4]. In this regard, different groups [5][6][7] exploited the integration of nanostructures with delivering capabilities with single-cell specificity and high throughput in biosensing platforms. These efforts provided powerful tools for advancing applications in therapeutics, diagnostics, and drug discovery, in order to reach an efficient and localized delivery on a chip. Despite these new tactics, there is still a critical need for the development of a functional approach that combines recording capabilities of nanostructured biosensors with intracellular delivery. The device should provide for tight contact between cells and electrode so as to enable highly localized delivery and optimal recording of action potentials in order to attain a high degree of prediction for the disease modeling and drug discovery. This \u201con-chip\u201d approach will help to gain deeper insight in several bio-related studies and analyses, providing a comprehensive knowledge of the entire cellular dynamics when selectively stimulated by the desired bio-molecules. In the first part of this dissertation, a solution will be proposed in order to fill this gap and respond to this need in the biology field. In the first chapter, I will describe briefly the principles of action potentials and how neurons and cardiomyocyte are composed, together with the development of electrophysiology and the advent of multielectrode arrays. In the second chapter, more details about fabrication and cell-electrode system modelling will be explained. In the same chapter, I will explore the development of multielectrode arrays up to the present days, along with the advent of nanotechnologies and the related techniques for improving the previous platforms. The different cell poration techniques will be described in order to reach the best recording capabilities without damaging cells. Electroporation, optoporation and spontaneous poration will be presented and the chosen technique for our application (electroporation) will be reviewed more in detail. In the third chapter, different methodologies for intracellular delivery will be explained, focusing also on the electroporation technique. A small paragraph about the integration of these techniques on chip will be inserted to illustrate the state of the art of these devices. The fourth chapter will explicate in details the Microfluidic multielectrode array idea, the approach used in order to fabricate this novel platform from scratch, the experiments carried out to verify its capabilities and the associated results. In the last paragraph, I will discuss how the proposed platform could became suitable for the day to day uses in research activity by employing nanoporous materials. In fact, big efforts are carried out in order to find appropriate metamaterials as substitutes of the 3D counterparts so as to decrease the cost of device manufacturing that makes them unfitting with research activity. As a novel electrode material, nanoporous metals possess unique properties, such as a low fabrication cost, high plasmonic enhancement and large surface-volume ratio[8]. Nanoporous gold behaves like a metamaterial whose effective dielectric response can be tuned accordingly to the wanted use. These properties make the material suitable for multiple biosensing application, from a high-performance and reliable SERS (surface enhanced raman scattering) substrate [9] to an electrode in CMOS MEAs capable of intracellular recordings[10]. All these properties were explored in the last years, but it could be interesting to further study if the characteristics of this material could make it a good photoelectrical modulating material for eliciting electrogenic cells firing activity. In this way, this technology could be in principle easily implemented on commercial CMOS devices, consenting stimulation and recording at single cell level with high-resolution sensors, opening the way to new methodologies for studying electrogenic cells and tissues. Electrical stimulation of excitable cells is the basis for many implantable devices in cardiac treatment and in neurological studies for treating debilitating neurological syndromes. In order to make the technique less invasive, optical stimulation was widely investigated [11]. The non-genetic photostimulation is starting to make its way in the field since it allows to avoid changing the biological framework by using transient thermal or electrochemical outputs from synthetic materials attached to the target cells[12]. If stimulated with impinging light these materials could inject free charges into the solution resulting in an ionic current at the interface able to eliciting of neurons[13] or cardiomyocyte action potentials. Plasmonic porous materials have all the suitable properties to be considered as an appealing tools for charge injection and consequently for stimulation of electrically active cells [14]. Thus, the second part of this dissertation will exploit the capabilities of these plasmonic metamaterials, placing particular emphasis on the possibility of photoelectrochemical modulation. In particular, in the fifth and last chapter I will describe all the properties and application of the porous material and the mechanism of photoemission. In the experimental paragraphs, the free charge photoemission properties of porous gold will be explored together with plasmonic non-genetic photostimulation of the cardiac cells on commercial CMOS MEAs

Neuron-Glial (NG) Interactions: A Microfluidic Examination of NG Emergent Responses for Repair

Neuron-glia communication is crucial to the development, plasticity, and repair of the nervous system (NS). While neurons are well known to conduct electrical impulses that transfer biological information and stimuli throughout the NS, our understanding of the roles of glia continues to evolve from when the cells were largely believed to act solely for neuronal support. Recent decades of research has shown that glia can alter metabolism, conduct impulses and change phenotype for NS repair. NG interactions have, thereby, become heavily researched in varied areas of biomedical engineering, including embryogenesis, neural regeneration, growth, and intracellular synaptic activity. However, while NG interactions are known to regulate survival, differentiation, communication, and targeted migration of neural cells, the molecular signals that orchestrate these behaviors remain incompletely understood. As a result, many emerging studies have embraced microfluidics to regulate the spatial and temporal stimuli delivered to neural cell groups and measure subsequent NG responses. The overall objective of this thesis was to examine emergent NG behavior in response to chemical stimuli within controlled microfluidic environments. Experiments examined NG behavior in the central and peripheral NS critical to neural repair. In the first model, we examined the behavior of transformed glial progenitors (in the form of Medulloblastoma (MB)), known to emulate developmental processes, to external stimuli using controlled microenvironments. We used a microfluidic system called the bridged mlane, which allows for steady-state, 1D, controllable concentration gradients along the length of its’ microchannel. The system was used to evaluate in vitro migratory responses of MB-derived cells to external signaling from Epidermal Growth Factor (EGF) and stromal cell-derived factor 1-alpha (SDF-1). Data demonstrated that MB cells exhibit dosage-dependent chemotaxis towards increasing concentration gradients. However, as glial behaviors are intricately linked with that of neuronal cells, we next used a more comprehensive neural model to examine the collective behavior of neural progenitors in response to chemotactic stimulation. Experiments examined the collective behavior of NG progenitor cell populations in response to stimulation via fibroblast growth factor (FGF) gradients using a developmental model of the central nervous system (CNS) in the Drosophila Melanogaster, 3rd instar larvae stage. Surprisingly, our data demonstrated that cells migrated larger distances and with higher directionality within collective groups of both neuronal and glial progenitors than in populations of glia only. Taken together, these results helped elucidate different modalities for directed movement that can be used for therapeutic techniques that leverage the interdependent NG relationship. The last model examined NG contributions to the formation of neuromuscular junctions (NMJ) in the peripheral nervous system (PNS). The glial component of the NMJ, the Schwann cells (SCs), are essential to NMJ development and function including remodeling and regeneration. SCs are critical for PNS regeneration, where studies have shown SC are able to trans-differentiate in order to create glial bridges that bypass non-functional neuronal nodes and isolate damaged neurons. However recent NMJ models mainly focus on motor neurons (MN) and muscle cells (MCs), some in vitro work has been utilized to study SCs, but their overall roles still remain to be well-defined and studied. To that end, the experiments used a compartmentalized microfluidic platform to demonstrate reproducible differentiation of skeletal myotubes with increased viability and length following the time-dependent addition of neuronal and glial cells. We lastly probed the guidance cues and migratory patterns of NGs towards various growth factors to elucidate emergent NMJ response. Our data illustrated there is a co-culture effect on receptor expression dependent on stimulation time. The data point to SCs as key players in stabilizing and maintaining in vitro NMJ models that will aid the development and testing of emerging therapies for neuromuscular dysfunction

Towards brain-on-a-chip:microfluidic and microelectrode array platforms for morphological and electrophysiological observations on the propagation of Alzheimer's disease

Since the first compartmentalized neuronal culture described by Robert B. Campenot in 1977, compartmentalized microfluidic devices have been widely used to engineer the cellular environment for cell culture. In previous research by Dr. Anja Kunze, a microfluidic device was able to build a "co-pathological" model with neuronal culture for neurodegenerative disease studies. In this model, two neuronal populations were cultured in independent compartments, while the axons of both populations were able to grow away from their own population and arrived in the same compartment, which was between the two compartments for the neuronal populations. When one neuronal population was exposed to a drug and expressed a specific disease state, different disease states were observed in the axon compartment towards the other unexposed cell population. This co-pathological pattern was achieved and early stage of disease propagation was observed in this compartmentalized microfluidic device. An example of this propagation pattern in the native brain is the well-known neurodegenerative disease Alzheimer's disease (AD), which still lacks effective treatments. In the AD brain, disease progression is observed from one brain region to another, eventually influencing the whole brain. This disease model can be mimicked in vitro within microdevices to assist neuroscientists in gaining a better understanding of the mechanisms of AD spreading in the native brain. In this thesis, we designed and fabricated compartmentalized microfluidic devices to build a co-pathological model to study the propagation of Tau pathology, which is one of the key pathological hallmarks of neurodegenerative disorders. Besides the morphological characteristics that we observe using our microfluidic device, microelectrode arrays (MEAs) technology, which is based on microtechnology and allows for recording extracellular neuronal activity, was integrated with the microfluidic device in this work. Together, the microfluidic and the integrated microfluidic-MEA devices provide us the possibility to monitor respectively time-variant morphological and electrophysiological alterations during disease spreading. We are therefore able to distinguish the contribution of neuron-to-neuron transmissions, observe different patterns of disease propagation with high and low drug-induced AD models, and observe the order in which the structural and functional alterations occur during AD progression. Based on the results that were achieved during our investigation of AD in this thesis, these microfluidic and integrated microdevices may potentially be used to study neurodegenerative diseases and perform pharmaceutical drug tests

Novel miniaturised and highly versatile biomechatronic platforms for the characterisation of melanoma cancer cells

There has been an increasing demand to acquire highly sensitive devices that are able to detect and characterize cancer at a single cell level. Despite the moderate progress in this field, the majority of approaches failed to reach cell characterization with optimal sensitivity and specificity. Accordingly, in this study highly sensitive, miniaturized-biomechatronic platforms have been modeled, designed, optimized, microfabricated, and characterized, which can be used to detect and differentiate various stages of melanoma cancer cells. The melanoma cell has been chosen as a legitimate cancer model, where electrophysiological and analytical expression of cell-membrane potential have been derived, and cellular contractile force has been obtained through a correlation with micromechanical deflections of a miniaturized cantilever beam. The main objectives of this study are in fourfold: (1) to quantify cell-membrane potential, (2) correlate cellular biophysics to respective contractile force of a cell in association with various stages of the melanoma disease, (3) examine the morphology of each stage of melanoma, and (4) arrive at a relation that would interrelate stage of the disease, cellular contractile force, and cellular electrophysiology based on conducted in vitro experimental findings. Various well-characterized melanoma cancer cell lines, with varying degrees of genetic complexities have been utilized. In this study, two-miniaturized-versatile-biomechatronic platforms have been developed to extract the electrophysiology of cells, and cellular mechanics (mechanobiology). The former platform consists of a microfluidic module, and stimulating and recording array of electrodes patterned on a glass substrate, forming multi-electrode arrays (MEAs), whereas the latter system consists of a microcantilever-based biosensor with an embedded Wheatstone bridge, and a microfluidic module. Furthermore, in support of this work main objectives, dedicated microelectronics together with customized software have been attained to functionalize, and empower the two-biomechatronic platforms. The bio-mechatronic system performance has been tested throughout a sufficient number of in vitro experiments.Open Acces

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

Neurochemical characterization of the rodent primary sensory system

Primary sensory neurons and their associated tissues are important targets for neurochemical study. Disorders of the sensory system, including chronic pain and itch, can be extremely devastating and, in many cases, difficult to treat. Part of the difficulty of treating such disorders is the limited understanding that we have for the multitude of chemical players involved in the communication of sensation within the nervous system. One particular set of intercellular signaling molecules, neuropeptides, are known to play an important role in the transmission of pain and itch signals from the peripheral system to the spinal cord. While we have a basic understanding of how many of these molecules are involved in sensory transmission, further knowledge would be benefited by more accurate and spatially relevant sampling and characterization. However, due to their low concentration and dynamic presence, the detection of these molecules in a non-targeted manner poses a unique challenge. This dissertation focuses on characterizing the peptides found in the tissues of the sensory system and released from primary sensory neurons in culture as well as improving culturing and stimulation paradigms for future research. We have worked to characterize the full content of peptides within the dorsal root ganglia, which houses the cell bodies of the primary sensory neurons, as well as other related tissues of rat and to detect changes in the peptide content of the dorsal root ganglia and dorsal horn upon generation of an itch model in mice. We have also designed a physiologically relevant sensory neuron culturing system and made strides toward spatially relevant release sampling and neuropeptide detection

Micro/nanofluidic and lab-on-a-chip devices for biomedical applications

Micro/Nanofluidic and lab-on-a-chip devices have been increasingly used in biomedical research [1]. Because of their adaptability, feasibility, and cost-efficiency, these devices can revolutionize the future of preclinical technologies. Furthermore, they allow insights into the performance and toxic effects of responsive drug delivery nanocarriers to be obtained, which consequently allow the shortcomings of two/three-dimensional static cultures and animal testing to be overcome and help to reduce drug development costs and time [2–4]. With the constant advancements in biomedical technology, the development of enhanced microfluidic devices has accelerated, and numerous models have been reported. Given the multidisciplinary of this Special Issue (SI), papers on different subjects were published making a total of 14 contributions, 10 original research papers, and 4 review papers. The review paper of Ko et al. [1] provides a comprehensive overview of the significant advancements in engineered organ-on-a-chip research in a general way while in the review presented by Kanabekova and colleagues [2], a thorough analysis of microphysiological platforms used for modeling liver diseases can be found. To get a summary of the numerical models of microfluidic organ-on-a-chip devices developed in recent years, the review presented by Carvalho et al. [5] can be read. On the other hand, Maia et al. [6] report a systematic review of the diagnosis methods developed for COVID-19, providing an overview of the advancements made since the start of the pandemic. In the following, a brief summary of the research papers published in this SI will be presented, with organs-on-a-chip, microfluidic devices for detection, and device optimization having been identified as the main topics.info:eu-repo/semantics/publishedVersio