Cell adhesion promotion strategies for signal transduction enhancement in microelectrode array in vitro electrophysiology: An introductory overview and critical discussion

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Microengineering The Neural Tube

Early embryonic development is a complex and highly regulated orchestra of instructive cues that collectively guide naïve stem cells towards progressively more specialized fates. In the neural tube, the precursor structure to the brain and spinal cord, these signals emanate from ‘organizing centers’ surrounding the neural tube. These organizing centers send out soluble cues or morphogens that diffuse tens to hundreds of microns to recipient cells residing in the neural tube. Re-creating this dynamic landscape of cues in vitro is impossible using standard cell culture tools and techniques. However, microfluidics is perfectly suited to fill this gap, allowing precise control over the microenvironment on the same length scale as the developing embryo. A microfluidic device is presented that is able to re-create some of the spatial patterning events that occur during the early development of the neural tube. This platform enables developmental biologists to reverse engineer development from the ground up, enabling researchers to pose radically new experiments to help answer some of the most relevant questions regarding fate specification in the developing neural tube. Here the device is used to guide mouse embryonic stem cells into motor neurons. Importantly, these motor neurons are able to be directed to differentiate in a defined region of the microdevice, a spatial patterning event that is the hallmark of the developing neural tube. For the first time it is now possible to study the effect of development cues on live populations of stem cells. The characterization of these fundamental developmental processes will prove invaluable in understanding how humans acquire both form and function. One day, it may allow researchers to harness these developmental techniques, which have been refined over thousands of years of evolution, to guide patient derived cells into any user defined cell fate

Glioma on Chips Analysis of glioma cell guidance and interaction in microfluidic-controlled microenvironment enabled by machine learning

In biosystems, chemical and physical fields established by gradients guide cell migration, which is a fundamental phenomenon underlying physiological and pathophysiological processes such as development, morphogenesis, wound healing, and cancer metastasis. Cells in the supportive tissue of the brain, glia, are electrically stimulated by the local field potentials from neuronal activities. How the electric field may influence glial cells is yet fully understood. Furthermore, the cancer of glia, glioma, is not only the most common type of brain cancer, but the high-grade form of it (glioblastoma) is particularly aggressive with cells migrating into the surrounding tissues (infiltration) and contribute to poor prognosis. In this thesis, I investigate how electric fields in the microenvironment can affect the migration of glioblastoma cells using a versatile microsystem I have developed. I employ a hybrid microfluidic design to combine poly(methylmethacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS), two of the most common materials for microfluidic fabrication. The advantages of the two materials can be complemented while disadvantages can be mitigated. The hybrid microfluidics have advantages such as versatile 3D layouts in PMMA, high dimensional accuracy in PDMS, and rapid prototype turnaround by facile bonding between PMMA and PDMS using a dual-energy double sided tape. To accurately analyze label-free cell migration, a machine learning software, Usiigaci, is developed to automatically segment, track, and analyze single cell movement and morphological changes under phase contrast microscopy. The hybrid microfluidic chip is then used to study the migration of glioblastoma cell models, T98G and U-251MG, in electric field (electrotaxis). The influence of extracellular matrix and chemical ligands on glioblastoma electrotaxis are investigated. I further test if voltage-gated calcium channels are involved in glioblastoma electrotaxis. The electrotaxes of glioblastoma cells are found to require optimal laminin extracellular matrices and depend on different types of voltage-gated calcium channels, voltage-gated potassium channels, and sodium transporters. A reversiblysealed hybrid microfluidic chip is developed to study how electric field and laminar shear can condition confluent endothelial cells and if the biomimetic conditions affect glioma cell adhesion to them. It is found that glioma/endothelial adhesion is mediated by the Ang1/Tie2 signaling axis and adhesion of glioma is slightly increased to endothelial cells conditioned with shear flow and moderate electric field. In conclusion, robust and versatile hybrid microsystems are employed for studying glioma biology with emphasis on cell migration. The hybrid microfluidic tools can enable us to elucidate fundamental mechanisms in the field of the tumor biology and regenerative medicine.Okinawa Institute of Science and Technology Graduate Universit

Micro- and Nanofluidics for Bionanoparticle Analysis

Bionanoparticles such as microorganisms and exosomes are recoganized as important targets for clinical applications, food safety, and environmental monitoring. Other nanoscale biological particles, includeing liposomes, micelles, and functionalized polymeric particles are widely used in nanomedicines. The recent deveopment of microfluidic and nanofluidic technologies has enabled the separation and anslysis of these species in a lab-on-a-chip platform, while there are still many challenges to address before these analytical tools can be adopted in practice. For example, the complex matrices within which these species reside in create a high background for their detection. Their small dimension and often low concentration demand creative strategies to amplify the sensing signal and enhance the detection speed. This Special Issue aims to recruit recent discoveries and developments of micro- and nanofluidic strategies for the processing and analysis of biological nanoparticles. The collection of papers will hopefully bring out more innovative ideas and fundamental insights to overcome the hurdles faced in the separation and detection of bionanoparticles

Development of novel tools for assisted reproductive technologies based on electrically switchable surfaces

A variety of stimuli have been explored in the last few decades to develop dynamic interfaces with biotechnological and biomedical applications, such as biosensors, point of care devices, cell behaviour control and tissue engineering. In this work, the use of an electrical stimulus was explored for the development of a smart switchable surface with the ability to, in an on-demand fashion, expose and conceal progesterone - an ovarian steroid hormone which plays a crucial role as a modulator of sperm function. In this system, an electric potential drives a conformational change in the surface bound peptide moiety with fast response time. Focus was given to the design of a device that could be used in assisted reproductive treatments and grown into a commercially marketable product. Whilst being developed for assessment of sperm quality and fertilizing potential, the application of this system can be widely extended as this approach can be applied to other relevant antigen-antibody systems, which have so far only been evaluated in static conditions. Fabrication of a micropatterned surface was performed and a novel method for orthogonal functionalisation of gold and glass was developed, where gold was functionalised with a polyethylene glycol thiol self-assembled monolayer (SAM) and glass was functionalised with a covalently bound poly-d-lysine layer for sperm cell attachment. In addition to the investigations on SAMs and mixed SAMs formed on gold, silicon and glass substrates, studies with fluospheres were also undertaken. These tools are aimed to be used for further studies with cells, namely the investigation of their response in terms of Ca2+ signalling, a key player in the regulation of sperm function

INTEGRATION OF CMOS TECHNOLOGY INTO LAB-ON-CHIP SYSTEMS APPLIED TO THE DEVELOPMENT OF A BIOELECTRONIC NOSE

This work addresses the development of a lab-on-a-chip (LOC) system for olfactory sensing. The method of sensing employed is cell-based, utilizing living cells to sense stimuli that are otherwise not easily sensed using conventional transduction techniques. Cells have evolved over millions of years to be exquisitely sensitive to their environment, with certain types of cells producing electrical signals in response to stimuli. The core device that is introduced here is comprised of living olfactory sensory neurons (OSNs) on top of a complementary metal-oxide-semiconductor (CMOS) integrated circuit (IC). This hybrid bioelectronic approach to sensing leverages the sensitivity of OSNs with the electronic signal processing capability of modern ICs. Intimately combining electronics with biology presents a number of unique challenges to integration that arise from the disparate requirements of the two separate domains. Fundamentally the obstacles arise from the facts that electronic devices are designed to work in dry environments while biology requires not only a wet environment, but also one that is precisely controlled and non-toxic. Design and modeling of such heterogeneously integrated systems is complicated by the lack of tools that can address the multiple domains and techniques required for integration, namely IC design, fluidics, packaging, and microfabrication, and cell culture. There also arises the issue of how to handle the vast amount of data that can be generated by such systems, and specifically how to efficiently identify signals of interest and communicate them off-chip. The primary contributions of this work are the development of a new packaging scheme for integration of CMOS ICs into fluidic LOC systems, a methodology for cross-coupled multi-domain iterative modeling of heterogeneously integrated systems, demonstration of a proof-of-concept bioelectronic olfactory sensor, and a novel event-based technique to minimize the bandwidth required to communicate the information contained in bio-potential signals produced by dense arrays of electrically active cells

Nanoimprint Lithography Technology and Applications

Nanoimprint Lithography (NIL) has been an interesting and growing field in recent years since its beginnings in the mid-1990s. During that time, nanoimprinting has undergone significant changes and developments and nowadays is a technology used in R&D labs and industrial production processes around the world. One of the exciting things about nanoimprinting process is its remarkable versatility and the broad range of applications. This reprint includes ten articles, which represent a small glimpse of the challenges and possibilities of this technology. Six contributions deal with nanoimprint processes aiming at specific applications, while the other four papers focus on more general aspects of nanoimprint processes or present novel materials. Several different types of nanoimprint processes are used: plate-to-plate, roll-to-plate, and roll-to-roll. Plate-to-plate NIL here also includes the use of soft and flexible stamps. The application fields in this reprint are broad and can be identified as plasmonics, superhydrophibicity, biomimetics, optics/datacom, and life sciences, showing the broad applicability of nanoimprinting. The sections on the nanoimprint process discuss filling and wetting aspects during nanoimprinting as well as materials for stamps and imprinting