A novel Three-Dimensional Micro-Electrode Array for in-vitro electrophysiological applications

Microelectrode arrays (MEAs) represent a powerful and popular tool to study in vitro neuronal networks and acute brain slices. The research standard for MEAs is planar or 2D-MEAs, which have been in existence for over 30 years and used for extracellular recording and stimulation from cultured neuronal cells and tissue slices. However, planar MEAs suffer from rapid data attenuation in the z-direction when stimulating/recording from 3D in-vitro neuronal cultures or brain slices. The existing proposed 3D in-vitro neuronal models allow to record the electrophysiological activity of the 3D network only from the bottom layer (i.e. the one directly coupled to the planar MEAs). Thus, to further develop and optimize such 3D neuronal network systems and to study and understand how the 3D neuronal network dynamics changes in different layers of the 3D structure, new three-dimensional microelectrodes arrays (3D-MEAs) are required. Early attempts in this field resulted in interesting integrated approaches toward protruding or spiked 3D-MEAs. Although these first prototypes could be successfully employed with brain slices, the limited heights of the electrodes (up to max 70 \u3bcm) and the peculiar shape of the recording areas made them not an ideal solution for 3D neuronal cultures. Moreover, a convenient and versatile method for the fabrication of multilevel 3D microelectrode arrays has yet to be obtained, due to the usually complicated and expensive designs and a lack of a full compatibility with standard MEAs both in terms of materials and recording area dimensions. To overcome the afore-mentioned challenges, in this work, I present the design, microfabrication, and characterization of a new 3D-MEA composed of pillar-shaped gold 3D structures with heights of more than 100 \u3bcm that can be used, in principle, on every kind of MEA, both custom-made and commercial. I successfully demonstrate the capability and ability of such 3D-MEA to record electrophysiological spontaneous activity from 3D engineered in-vitro neuronal networks and both 4-AP-induced epileptiform-like and electrically-evoked activity from mouse acute brain slices. I also demonstrate how the developed 3D-MEA allows better recording and stimulating conditions while interfacing with acute brain slices as compared to planar electrode arrays and previously reported 3D MEA technologies

Carbon Fiber Microelectrode Arrays for Neuroprosthetic and Neuroscience Applications.

The aim of this work is to develop, validate, and characterize the insertion mechanism, tissue response, and recording longevity of a new high-density carbon fiber microelectrode array. This technology was designed to significantly improve the field of penetrating microelectrodes while simultaneously accommodating the variable needs of both neuroscientists and neural engineers. The first study presents the fabrication and insertion dynamics of a high-density carbon fiber electrode array using a dual sided printed circuit board platform. The use of this platform has pushed electrode density to limits not seen in other works. This necessitated the use of an encapsulation method that served to temporarily stiffen the fibers during insertion, but did not enter the brain as many other shuttles do for other probe designs. The initial findings in this work informed the development of an even higher density array using a silicon support structure as a backbone. The second study reports on the tissue reaction of chronically implanted carbon fiber electrode arrays as compared to silicon electrodes. Due to their smaller footprint, the reactive response to carbon fibers should be greatly attenuated, if not non-existent. Results show a scarring response to the implanted silicon electrode with elevated astrocyte and microglia activity coupled to a local decrease in neuronal density. The area implanted with the carbon fiber electrodes showed a varied response, from no detectable increase in astrocytic or microglial activity to an elevated activation of both cell types, but with no detectable scars. Neuronal density in the carbon fiber implant region was unaffected. The data demonstrates that the small carbon fiber profile, even in an array configuration, shows an attenuated reactive response with no visible scaring. The final study reports on the viability of chronically implanted high-density carbon fiber arrays as compared to more traditional silicon planar arrays with comparable site sizes. While most new probe technologies or designs are able to demonstrate proof of concept functionality in acute preparations, very few show the ability to record chronic unit activity. This study aims to provide a comprehensive analysis of electrophysiology data collected over implant durations ranging from 3 – 5 months.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111557/1/parasp_1.pd

The potential of microelectrode arrays and microelectronics for biomedical research and diagnostics

Planar microelectrode arrays (MEAs) are devices that can be used in biomedical and basic in vitro research to provide extracellular electrophysiological information about biological systems at high spatial and temporal resolution. Complementary metal oxide semiconductor (CMOS) is a technology with which MEAs can be produced on a microscale featuring high spatial resolution and excellent signal-to-noise characteristics. CMOS MEAs are specialized for the analysis of complete electrogenic cellular networks at the cellular or subcellular level in dissociated cultures, organotypic cultures, and acute tissue slices; they can also function as biosensors to detect biochemical events. Models of disease or the response of cellular networks to pharmacological compounds can be studied in vitro, allowing one to investigate pathologies, such as cardiac arrhythmias, memory impairment due to Alzheimer's disease, or vision impairment caused by ganglion cell degeneration in the retin

Micromachined three-dimensional electrode arrays for in-vitro and in-vivo electrogenic cellular networks

This dissertation presents an investigation of micromachined three-dimensional microelectrode arrays (3-D MEAs) targeted toward in-vitro and in-vivo biomedical applications. Current 3-D MEAs are predominantly silicon-based, fabricated in a planar fashion, and are assembled to achieve a true 3-D form: a technique that cannot be extended to micro-manufacturing. The integrated 3-D MEAs developed in this work are polymer-based and thus offer potential for large-scale, high volume manufacturing. Two different techniques are developed for microfabrication of these MEAs - laser micromachining of a conformally deposited polymer on a non-planar surface to create 3-D molds for metal electrodeposition; and metal transfer micromolding, where functional metal layers are transferred from one polymer to another during the process of micromolding thus eliminating the need for complex and non-repeatable 3-D lithography processes. In-vitro and in-vivo 3-D MEAs are microfabricated using these techniques and are packaged utilizing Printed Circuit Boards (PCB) or other low-cost manufacturing techniques. To demonstrate in-vitro applications, growth of 3-D co-cultures of neurons/astrocytes and tissue-slice electrophysiology with brain tissue of rat pups were implemented. To demonstrate in-vivo application, measurements of nerve conduction were implemented. Microelectrode impedance models, noise models and various process models were evaluated. The results confirmed biocompatibility of the polymers involved, acceptable impedance range and noise of the microelectrodes, and potential to improve upon an archaic clinical diagnostic application utilizing these 3-D MEAs.Ph.D.Committee Chair: Mark G. Allen; Committee Member: Elliot L. Chaikof; Committee Member: Ionnis (John) Papapolymerou; Committee Member: Maysam Ghovanloo; Committee Member: Oliver Bran

Substrate arrays of iridium oxide microelectrodes for in vitro neuronal interfacing

The design of novel bidirectional interfaces for in vivo and in vitro nervous systems is an important step towards future functional neuroprosthetics. Small electrodes, structures and devices are necessary to achieve high-resolution and target-selectivity during stimulation and recording of neuronal networks, while significant charge transfer and large signal-to-noise ratio are required for accurate time resolution. In addition, the physical properties of the interface should remain stable across time, especially when chronic in vivo applications or in vitro long-term studies are considered, unless a procedure to actively compensate for degradation is provided. In this short report, we describe the use and fabrication of arrays of 120 planar microelectrodes (MEAs) of sputtered Iridium Oxide (IrOx). The effective surface area of individual microelectrodes is significantly increased using electrochemical activation, a procedure that may also be employed to restore the properties of the electrodes as required. The electrode activation results in a very low interface impedance, especially in the lower frequency domain, which was characterized by impedance spectroscopy. The increase in the roughness of the microelectrodes surface was imaged using digital holographic microscopy and electron microscopy. Aging of the activated electrodes was also investigated, comparing storage in saline with storage in air. Demonstration of concept was achieved by recording multiple single-unit spike activity in acute brain slice preparations of rat neocortex. Data suggests that extracellular recording of action potentials can be achieved with planar IrOx MEAs with good signal-to-noise ratios. © 2009 Gawad, Giugliano, Heuschkel, Wessling, Markram, Schnakenberg, Renaud and Morgan

Master of Science

thesisOver the past four decades, Multielectrode Array (MEA) devices have played a major role in electrophysiology by providing a simpler solution to simultaneous multi-site chronic extracellular recording: in vivo and in vitro. While a wide range of devices have been developed, almost all of them are limited to culturing and recording from one cell type, in vitro; and tissue surfaces, in vivo and in vitro. Most tissues are formed by different cell types that interact to maintain tissue function, like the heart which is composed mainly of cardio-myocytes and fibroblasts. Direct recording from such organs usually employs plunge-type electrodes which induce tissue damage and require better handling for sustenance. To better understand the functioning of such tissues, it is imperative to utilize recording systems that allow interactions between two or more cell types and at the same time sustain cultures with controlled cell number and distribution. In this thesis, the design, fabrication process, and characterization of an MEA device called the PerFlexMEA (Perforated Flexible MEA) is presented. It enables the generation and sustenance of a preparation with two cell types while recording their electrical activity. PerFlexMEA was developed using a thin (9?m) perforated Polycarbonate Track Etch (PCTE) membrane (3?m diam. pores, 200,000 pores/cm2) as substrate where cells can be cultured on both sides, allowing gap junction formation across the membrane via the pores. Cell number and distribution can be controlled on either side. The PerFlexMEA comprises a 4 × 5 array of square gold electrodes, each measuring 50 ?m × 50 ?m spaced 500 ?m apart. Parylene was patterned to insulate the leads (50 ?m thick) connecting the recording electrodes to the contact pads. A coinshaped device was designed to house the PerFlexMEA and to insulate its cell culture zone (wet) from contact pads (dry). Cardiomyocytes, isolated from neonatal mice were plated on the recording side of PerFlexMEA and electrical activity was recorded at a signal to noise ratio of 8.6 and peak to peak voltage of 200 ?V

Intracranial neuronal ensemble recordings and analysis in epilepsy

Pathological neuronal firing was demonstrated 50 years ago as the hallmark of epileptically transformed cortex with the use of implanted microelectrodes. Since then, microelectrodes remained only experimental tools in humans to detect unitary neuronal activity to reveal physiological and pathological brain functions. This recording technique has evolved substantially in the past few decades; however, based on recent human data implying their usefulness as diagnostic tools, we expect a substantial increase in the development of microelectrodes in the near future. Here, we review the technological background and history of microelectrode array development for human examinations in epilepsy, including discussions on of wire-based and microelectrode arrays fabricated using micro-electro-mechanical system (MEMS) techniques and novel future techniques to record neuronal ensemble. We give an overview of clinical and surgical considerations, and try to provide a list of probes on the market with their availability for human recording. Then finally, we briefly review the literature on modulation of single neuron for the treatment of epilepsy, and highlight the current topics under examination that can be background for the future development

Development of Epilepsy-on-a-chip System for High-throughput Antiepileptogenic Drug Discovery

Epilepsy is one of the most common neurological disorders and affects millions of people in the United States. Currently available antiepileptic drugs require continuous administration for suppression of seizures and have not been shown to prevent the development of epilepsy (epileptogenesis). The discovery of antiepileptogenic drug is complicated by the long time course of epileptogenesis in animal models of epilepsy and the requirement of continuous monitoring of epileptiform activity in vivo for the assessment of drug efficacy. In recent years, organotypic hippocampal cultures have been increasingly used as an in vitro model of post-traumatic epilepsy in both basic and translational research. Epileptogenesis in this in vitro model has a compressed time scale and can be monitored by detection of electrographic and biochemical markers of seizure-like activity. However, the lack of a scalable chronic electrical recording platform is a significant bottleneck in high-throughput antiepileptogenic drug discovery using organotypic cultures.In an effort to circumvent the throughput limitations of in vitro antiepileptogenic drug discovery, a hybrid microfluidic-multiple electrode array (µflow-MEA) technology was developed for scalable chronic electrical assay of epileptogenesis in vitro. Specifically, the microfluidic perfusion technique was utilized to miniature the culture platform, which enabled the long-term maintenance of an organotypic culture array on a single device. The integration of the microfluidic perfusion system with a customized planar MEA allowed for parallel continuous recordings. As a proof-of-concept demonstration, a pilot screen of receptor tyrosine kinase (RTK) inhibitor library was performed on µflow-MEA based electrical assay platform. The screen results revealed significant antiepileptogenic effect of cFMS RTK inhibitor.This thesis also provides further validation of the organotypic hippocampal culture model of epilepsy by investigating the influence of culture medium composition on epileptogenesis. We found that epileptogenesis occurred in any culture medium that was capable of supporting neural survival, indicating that culture medium composition has limited influence on epileptogenesis in organotypic hippocampal cultures.It is hoped that the techniques presented in this thesis will accelerate the antiepileptogenic drug discovery and contribute to the development of new therapeutics to treat individuals at risk of epileptogenesis