Interaction champ électrique cellule (conception de puces microfluidiques pour l'appariement cellulaire et la fusion par champ électrique pulsé)

La fusion cellulaire est une méthode de génération de cellules hybrides combinant les propriétés spécifiques des cellules mères. Initialement développée pour la production d anticorps, elle est maintenant aussi investiguée pour l immunothérapie du cancer. L électrofusion consiste à produire ces hybrides en utilisant un champ électrique pulsé. Cette technique présente de meilleurs rendements que les fusions chimiques ou virales, sans introduire de contaminant. L électrofusion est actuellement investiguée en cuve d électroporation où le champ électrique n est pas contrôlable avec précision et le placement cellulaire impossible, produisant de faibles rendements binucléaires. Afin d augmenter le rendement et la qualité de fusion, la capture et l appariement des cellules s avèrent alors nécessaires.Notre objectif a été de développer et de réaliser des biopuces intégrant des microélectrodes et des canaux microfluidiques afin de positionner et d apparier les cellules avant leur électrofusion. Une première structure de piégeage se basant sur des plots isolants et l utilisation de la diélectrophorèse a été réalisée. Afin d effectuer des expérimentations sous flux, une méthode de scellement des canaux, biocompatible et étanche a été développée. Puis, le milieu d expérimentation a été adapté pour l électrofusion. En confrontant les résultats des expériences biologiques aux simulations numériques, nous avons pu démontrer que l application d impulsions électriques induisait la diminution de la conductivité cytoplasmique. Nous avons ensuite validé la structure par l électrofusion de cellules. Un rendement de 55% avec une durée de fusion membranaire de 6 s a été obtenu. Dans un second temps, nous avons proposé deux microstructures de piégeage pour l électrofusion haute densité. La première se base sur un piégeage fluidique, alors que la seconde, utilise ladiélectrophorèse sans adressage électrique à l aide de plots conducteurs. Jusqu à 75% des cellules fusionnent dans cette dernière structure. Plus de 97% des hybridomes produits sont binucléaires. Le piégeage étant réversible, les hybridomes peuvent ensuite être collectés pour des études ultérieures.Cell fusion is a method to generate a hybrid cell combing the specific properties of its progenitor cells. Initially developed for antibody production, it is now also investigated for cancer immunotherapy. Electrofusion consists on the production of hybridoma using electric pulses. Compared to viral or chemical methods, electrofusion shows higher yields and this system is contaminant free. Actually, electrofusion is investigated in electroporation cuvettes, where the electric field is not precisely controllable and cell placement impossible, resulting in low binuclear hibridoma yields. To improve the fusion quality and yield, cell capture and pairing are necessary.Our objective was the development and realization of biochips involving microelectrodes and microfluidic channels to place and pair cells prior to electrofusion. A first trapping structure based on insulators and the use of dielectrophoresis has been achieved. In order to perform fluidic experiments, a biocompatible irreversible packaging was developed. Then, the experimental medium was optimized for electrofusion. Confronting the biological experiments and the numerical simulations, we showed that the application of electric pulses leads to a decrease of the cytoplasmic conductivity. The microstructure was validated by cell electrofusion. A yield of 55%, with a membrane fusion duration of 6 s has been achieved. Secondly, we proposed two trapping microstructures for high density electrofusions. The first one is based on a fluidic trapping while the second one uses dielectrophoresis, free of electric wiring, thanks to conductive pads. Up to 75% of paired cells were successfully electrofused with the conductive pads. More than 97% of the hybridoma were binuclear. The trapping being reversible, the hybridoma can be collected for further analysis.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

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

High aspect ratio electrodes for high yield electroporation of cells

Electroporation is a widely used process in cell biology studies. It uses an electric field to create pores on the cell membrane in order to either insert exogenous molecules inside the cells or disrupt the cell membrane to kill the cells. Current micro-fluidic electroporation devices use the planar electrodes situated at the bottom of a microchannel. These planar electrodes i) require a high voltage and ii) generate a nonuniform electric field which result in low yield of the electroporation. The standard silicon microfabrication technologies are not suitable to fabricate non-planar electrodes required to increase the yield of electroporation. In this research, an electroporation device is fabricated with an array of five pairs of three dimensional (3D) electrodes situated along the sidewalls of a microchannel. These 3D electrodes are fabricated by filling the molten indium inside the chosen microchannels. The indium filling method allows the fabrication of microstructures with planar dimensions larger than ~30 µm regardless of their height, integrated into the PDMS device. The selective electroporation of fibroblast cells is successfully demonstrated using a fabricated device by applying a low voltage (1.67 V). The uniform electric field generated in cross sections of microchannel by 3D electrodes will avoid the limitations of planar electrodes by i) preventing cell death due to an excessive electric field and ii) preventing lack of electroporation due to a low electric field. As a result, these 3D electrodes should be capable of increasing the yield of electroporation

An integrated chip-based device for droplet-flow polymerase chain reaction

The polymerase chain reaction (PCR) is an important in-vitro technique in molecular biology for amplifying trace quantities of deoxyribonucleic acid (DNA). PCR is carried out by mixing the DNA molecules to be amplified with primers, polymerase enzymes and deoxynucleotide triphosphates (dNTPs) in a suitable buffer solution. A conventional thermal-cycler is then used to cycle the PCR mixture between multiple temperatures for denaturation, annealing and extension. Bench-top thermal cyclers have large thermal masses and use large sample volumes, leading to overly long cycling times, excessive energy and material consumption, inhomogeneity in the reaction environment, and an inability to handle large numbers of small volume aliquots. Microfluidic technologies overcome many of the limitations of bench-top thermal cyclers, providing a more controlled approach to PCR. Droplet-flow is one of the most promising microfluidic methods for carrying out PCR. The droplet-flow approach uses small water-in-oil droplets for compartmentalisation of the PCR reaction mixture, with each droplet behaving like an individual reaction chamber. By flowing the droplets over different temperatures for denaturation, annealing and extension, rapid thermal cycling can be achieved, greatly reducing the reaction time relative to bench-top thermal cyclers. The use of an oil phase to encapsulate the aqueous PCR mixture as droplets also prevents unwanted surface interactions and flow dispersion that can adversely affect the PCR yield. Here we describe an integrated microfluidic device for carrying out droplet-flow PCR. Instead of using multiple temperature zones to thermally-cycle the flowing droplets, the device used an on-chip radial temperature gradient. The droplets passed through microchannels arranged in a spoke-like geometry, causing them to pass backwards and forwards along the radial temperature gradient and so undergo the repeated thermal cycling required for PCR. The device reported here builds on an earlier plastic microfluidic PCR device (by Schaerli et al.) in which the radial temperature gradient was generated using a bulky external heater and a thermoelectric cooler, together with heat sinks and fans. In the silicon- and glass-based device reported here, integrated heaters, temperature sensors and air gaps (for passive cooling) were used to generate the temperature gradient, leading to significant miniaturisation of the device. The dimensions of the complete device assembly were 6.0 cm x 5.0 cm x 2.0 cm compared to 25.0 cm x 25.0 cm x 25.0 cm for the device by Schaerli et al.. Despite the small size of the device, the achievable temperature gradient on the chip was sizeable. For instance, when the central heater was set to 92.0 °C, the temperature at the periphery was ~60.0 °C, corresponding to a temperature difference of ~32.0 °C – easily sufficient for PCR applications. Using chemical modification, the hydrophilic walls of the microchannel were rendered hydrophobic. An on-chip T-junction or flow-focusing junction was subsequently used to merge the oil and aqueous streams to generate the PCR-containing water-in-oil droplets. A PCR recipe was optimised on a bench-top thermal cycle. With this recipe, droplet-flow PCR was conducted on the PCR device by flowing the generated droplets up and down the radial temperature gradient to induce thermal cycling. Gel electrophoresis analysis of the collected droplets from the device showed the presence of the PCR product, confirming the ability of the integrated device to conduct droplet-flow PCR. By varying the central temperature of the PCR device and the flow rate of the droplets, the yield of the PCR product could be tuned. By serially diluting the concentration of the DNA molecules, it was found that the PCR device was able to amplify concentrations as low as 0.01 pM to a level detectable by gel electrophoresis. When coupled to a laser-induced fluorescence detection system, the emission from the PCR mixture in the water-in-oil droplets could be successfully detected for each PCR cycle. The increase in the fluorescence over successive PCR cycles once again verified the feasibility of carrying out droplet-flow PCR on the integrated device.Open Acces

Microfluidic and Electrokinetic Manipulation of Single Cells

Traditional cell assays report on the average results of a cell population. However, a wide range of new tools are being developed for a fundamental understanding of single cell's functionality. Nonetheless, the current tools are either limited in their throughput or the accuracy of the analysis. One such technology is electrorotation. Although it is known to be unique in its capability for single-cell characterization, it is commonly a slow technique with a processing time of about 30 minutes per cell. For this reason, this thesis focuses on the development of a 3D electrode based electrorotation setup for fast and automatic extraction of a single cell's spectrum. For this purpose, new fabrication processes for 3D electrodes were developed to achieve high-resolution patterning of 3D metal electrodes. The first process we developed was a subtractive one based on passivated silicon structures and the second process was an additive one based on SU-8 photolithography. The additive nature of the second process enables high patterning resolution of electrodes and connection layers, while providing high conductivity thanks to the use of standard metal films. The electrodes have been characterized by different electrical measurements to ensure a proper connection and side-wall exposure. Furthermore, we characterized and compared the sheet resistance of planar and vertical layers. A further microfabrication process was developed for integrating the electrodes into microfluidic channels. The process was designed to enable the use of high numerical aperture lenses; for that purpose, a PDMS-mediated bonding process was engineered to seal the channels with a thin glass coverslip. Moreover, the development of a process to realize microfluidic access holes on the back of the wafer reduces the footprint of the chips and facilitates access for the microscope optics. Finally, a pressure-driven system was used together with the chips to achieve high control of liquid injections and to enable fast and precise flow stop. The combination of such a system, together with the dielectrophoretic forces that can be applied by the 3D electrodes, allows accurate positioning of single cells inside the 3D electrode quadrupole. The particles can then be analyzed by electrorotation. For this purpose, a custom Labview interface was built to coordinate the full setup and to acquire a full electrorotation spectrum in less than 3 minutes

소형동물의 뇌신경 자극을 위한 완전 이식형 신경자극기

학위논문(박사)--서울대학교 대학원 :공과대학 전기·정보공학부,2020. 2. 김성준.In this study, a fully implantable neural stimulator that is designed to stimulate the brain in the small animal is described. Electrical stimulation of the small animal is applicable to pre-clinical study, and behavior study for neuroscience research, etc. Especially, behavior study of the freely moving animal is useful to observe the modulation of sensory and motor functions by the stimulation. It involves conditioning animal's movement response through directional neural stimulation on the region of interest. The main technique that enables such applications is the development of an implantable neural stimulator. Implantable neural stimulator is used to modulate the behavior of the animal, while it ensures the free movement of the animals. Therefore, stable operation in vivo and device size are important issues in the design of implantable neural stimulators. Conventional neural stimulators for brain stimulation of small animal are comprised of electrodes implanted in the brain and a pulse generation circuit mounted on the back of the animal. The electrical stimulation generated from the circuit is conveyed to the target region by the electrodes wire-connected with the circuit. The devices are powered by a large battery, and controlled by a microcontroller unit. While it represents a simple approach, it is subject to various potential risks including short operation time, infection at the wound, mechanical failure of the device, and animals being hindered to move naturally, etc. A neural stimulator that is miniaturized, fully implantable, low-powered, and capable of wireless communication is required. In this dissertation, a fully implantable stimulator with remote controllability, compact size, and minimal power consumption is suggested for freely moving animal application. The stimulator consists of modular units of surface-type and depth-type arrays for accessing target brain area, package for accommodating the stimulating electronics all of which are assembled after independent fabrication and implantation using customized flat cables and connectors. The electronics in the package contains ZigBee telemetry for low-power wireless communication, inductive link for recharging lithium battery, and an ASIC that generates biphasic pulse for neural stimulation. A dual-mode power-saving scheme with a duty cycling was applied to minimize the power consumption. All modules were packaged using liquid crystal polymer (LCP) to avoid any chemical reaction after implantation. To evaluate the fabricated stimulator, wireless operation test was conducted. Signal-to-Noise Ratio (SNR) of the ZigBee telemetry were measured, and its communication range and data streaming capacity were tested. The amount of power delivered during the charging session depending on the coil distance was measured. After the evaluation of the device functionality, the stimulator was implanted into rats to train the animals to turn to the left (or right) following a directional cue applied to the barrel cortex. Functionality of the device was also demonstrated in a three-dimensional maze structure, by guiding the rats to navigate better in the maze. Finally, several aspects of the fabricated device were discussed further.본 연구에서는 소형 동물의 두뇌를 자극하기 위한 완전 이식형 신경자극기가 개발되었다. 소형 동물의 전기자극은 전임상 연구, 신경과학 연구를 위한 행동연구 등에 활용된다. 특히, 자유롭게 움직이는 동물을 대상으로 한 행동 연구는 자극에 의한 감각 및 운동 기능의 조절을 관찰하는 데 유용하게 활용된다. 행동 연구는 두뇌의 특정 관심 영역을 직접적으로 자극하여 동물의 행동반응을 조건화하는 방식으로 수행된다. 이러한 적용을 가능케 하는 핵심기술은 이식형 신경자극기의 개발이다. 이식형 신경자극기는 동물의 움직임을 방해하지 않으면서도 그 행동을 조절하기 위해 사용된다. 따라서 동물 내에서의 안정적인 동작과 장치의 크기가 이식형 신경자극기를 설계함에 있어 중요한 문제이다. 기존의 신경자극기는 두뇌에 이식되는 전극 부분과, 동물의 등 부분에 위치한 회로부분으로 구성된다. 회로에서 생산된 전기자극은 회로와 전선으로 연결된 전극을 통해 목표 지점으로 전달된다. 장치는 배터리에 의해 구동되며, 내장된 마이크로 컨트롤러에 의해 제어된다. 이는 쉽고 간단한 접근방식이지만, 짧은 동작시간, 이식부위의 감염이나 장치의 기계적 결함, 그리고 동물의 자연스러운 움직임 방해 등 여러 문제점을 야기할 수 있다. 이러한 문제의 개선을 위해 무선통신이 가능하고, 저전력, 소형화된 완전 이식형 신경자극기의 설계가 필요하다. 본 연구에서는 자유롭게 움직이는 동물에 적용하기 위하여 원격 제어가 가능하며, 크기가 작고, 소모전력이 최소화된 완전이식형 자극기를 제시한다. 설계된 신경자극기는 목표로 하는 두뇌 영역에 접근할 수 있는 표면형 전극과 탐침형 전극, 그리고 자극 펄스 생성 회로를 포함하는 패키지 등의 모듈들로 구성되며, 각각의 모듈은 독립적으로 제작되어 동물에 이식된 뒤 케이블과 커넥터로 연결된다. 패키지 내부의 회로는 저전력 무선통신을 위한 지그비 트랜시버, 리튬 배터리의 재충전을 위한 인덕티브 링크, 그리고 신경자극을 위한 이상성 자극파형을 생성하는 ASIC으로 구성된다. 전력 절감을 위해 두 개의 모드를 통해 사용률을 조절하는 방식이 장치에 적용된다. 모든 모듈들은 이식 후의 생물학적, 화학적 안정성을 위해 액정 폴리머로 패키징되었다. 제작된 신경자극기를 평가하기 위해 무선 동작 테스트가 수행되었다. 지그비 통신의 신호 대 잡음비가 측정되었으며, 해당 통신의 동작거리 및 데이터 스트리밍 성능이 검사되었고, 장치의 충전이 수행될 때 코일간의 거리에 따라 전송되는 전력의 크기가 측정되었다. 장치의 평가 이후, 신경자극기는 쥐에 이식되었으며, 해당 동물은 이식된 장치를 이용해 방향 신호에 따라 좌우로 이동하도록 훈련되었다. 또한, 3차원 미로 구조에서 쥐의 이동방향을 유도하는 실험을 통하여 장치의 기능성을 추가적으로 검증하였다. 마지막으로, 제작된 장치의 특징이 여러 측면에서 심층적으로 논의되었다.Chapter 1 : Introduction 1 1.1. Neural Interface 2 1.1.1. Concept 2 1.1.2. Major Approaches 3 1.2. Neural Stimulator for Animal Brain Stimulation 5 1.2.1. Concept 5 1.2.2. Neural Stimulator for Freely Moving Small Animal 7 1.3. Suggested Approaches 8 1.3.1. Wireless Communication 8 1.3.2. Power Management 9 1.3.2.1. Wireless Power Transmission 10 1.3.2.2. Energy Harvesting 11 1.3.3. Full implantation 14 1.3.3.1. Polymer Packaging 14 1.3.3.2. Modular Configuration 16 1.4. Objectives of This Dissertation 16 Chapter 2 : Methods 18 2.1. Overview 19 2.1.1. Circuit Description 20 2.1.1.1. Pulse Generator ASIC 21 2.1.1.2. ZigBee Transceiver 23 2.1.1.3. Inductive Link 24 2.1.1.4. Energy Harvester 25 2.1.1.5. Surrounding Circuitries 26 2.1.2. Software Description 27 2.2. Antenna Design 29 2.2.1. RF Antenna 30 2.2.1.1. Design of Monopole Antenna 31 2.2.1.2. FEM Simulation 31 2.2.2. Inductive Link 36 2.2.2.1. Design of Coil Antenna 36 2.2.2.2. FEM Simulation 38 2.3. Device Fabrication 41 2.3.1. Circuit Assembly 41 2.3.2. Packaging 42 2.3.3. Electrode, Feedthrough, Cable, and Connector 43 2.4. Evaluations 45 2.4.1. Wireless Operation Test 46 2.4.1.1. Signal-to-Noise Ratio (SNR) Measurement 46 2.4.1.2. Communication Range Test 47 2.4.1.3. Device Operation Monitoring Test 48 2.4.2. Wireless Power Transmission 49 2.4.3. Electrochemical Measurements In Vitro 50 2.4.4. Animal Testing In Vivo 52 Chapter 3 : Results 57 3.1. Fabricated System 58 3.2. Wireless Operation Test 59 3.2.1. Signal-to-Noise Ratio Measurement 59 3.2.2. Communication Range Test 61 3.2.3. Device Operation Monitoring Test 62 3.3. Wireless Power Transmission 64 3.4. Electrochemical Measurements In Vitro 65 3.5. Animal Testing In Vivo 67 Chapter 4 : Discussion 73 4.1. Comparison with Conventional Devices 74 4.2. Safety of Device Operation 76 4.2.1. Safe Electrical Stimulation 76 4.2.2. Safe Wireless Power Transmission 80 4.3. Potential Applications 84 4.4. Opportunities for Further Improvements 86 4.4.1. Weight and Size 86 4.4.2. Long-Term Reliability 93 Chapter 5 : Conclusion 96 Reference 98 Appendix - Liquid Crystal Polymer (LCP) -Based Spinal Cord Stimulator 107 국문 초록 138 감사의 글 140Docto

Versatile dielectrophoresis based microfluidic platforms for chemical stimulation of cells

The purpose of this project is to develop versatile microfluidic systems, which take advantage of dielectrophoresis, for the rapid creation of customized cell clusters, chemical stimulation of the patterned cells under well-controlled environmental conditions, and analysis of cellular responses using different microscopic techniques. As the first contribution, the author shows that the reorientation of the microfluidic channel with respect to the microelectrodes can be utilized to alter the characteristics of the dielectrophoretic (DEP) system. This enables the author to change the location and density of immobilized viable cells across the channel, release viable cells along customized numbers of streams within the channel, and improve the sorting of viable and nonviable cells in terms of flow throughput and efficiency of the system. As the second contribution, the author presents a novel approach to change the DEP response of nonviable yeast cells by chemically altering their surface properties. The author’s studies show that treating nonviable yeast cells with low concentrations of ionic surfactants can significantly change their surface properties, making them exhibit a strong positive DEP response, even at high medium conductivities. The capability of this treatment is demonstrated in two proof-of-concept experiments to create isolated or adjacent clusters of viable and nonviable cells next to each other. As the third contribution, the author utilizes dielectrophoresis for studying the dynamic response of cells following chemical stimulation. The DEP system enables separation of the budding yeasts from a background of non-budding cells, and their subsequent immobilization onto the microelectrodes at desired densities. The immobilized yeasts are then stimulated with Lyticase to remove the cell wall and convert them into spheroplasts in a dynamic process, which depends on the concentration of Lyticase. As the fourth contribution, the author introduces a novel method for immobilization of the cell organelles released from the lysed cells by patterning multi-walled carbon nanotubes (MWCNTs) between the microelectrodes. A strong electric field can be induced at the free ends of MWCNT chains, which is utilized to immobilize the released cell organelles from budding yeast cells after treating them with high concentration of Lyticase. As the fifth contribution, the author develops a DEP-based microfluidic platform for interfacing non-adherent cells with high-resolution scanning electron microscopy (SEM). The developed DEP system enables rapid immobilization, on-chip chemical stimulation and fixation, and dehydration of samples without deposition of chemical residues over the cell surface. These advantages are demonstrated for comparing the morphological changes of non-budding and budding yeast cells following Lyticase treatment. In summary, the research conducted by the PhD candidate enables studying of the dynamic cell responses under various chemical treatments using versatile DEP based microfluidic platforms. The PhD candidate also believes that the presented research will offer practical solutions for future biomedical micro-devices

A microgripper for single cell manipulation

This thesis presents the development of an electrothermally actuated microgripper for the manipulation of cells and other biological particles. The microgripper has been fabricated using a combination of surface and bulk micromachining techniques in a three mask process. All of the fabrication details have been chosen to enable a tri-layer, polymer (SU8) - metal (Au) - polymer (SU8), membrane to be released from the substrate stress free and without the need for sacrificial layers. An actuator design, which completely eliminates the parasitic resistance of the cold arm, is presented. When compared to standard U-shaped actuators, it improves the thermal efficiency threefold. This enables larger displacements at lower voltages and temperatures. The microgripper is demonstrated in three different configurations: normally open mode, normally closed mode, and normally open/closed mode. It has-been modelled using two coupled analytical models - electrothermal and thermomechanical - which have been custom developed for this application. Unlike previously reported models, the electrothermal model presented here includes the heat exchange between hot and cold arms of the actuators that are separated by a small air gap. A detailed electrothermomechanical characterisation of selected devices has permitted the validation of the models (also performed using finite element analysis) and the assessment of device performance. The device testing includes electrical, deflection, and temperature measurements using infrared (IR) thermography, its use in polymeric actuators reported here for the first time. Successful manipulation experiments have been conducted in both air and liquid environments. Manipulation of live cells (mice oocytes) in a standard biomanipulation station has validated the microgripper as a complementary and unique tool for the single cell experiments that are to be conducted by future generations of biologists in the areas of human reproduction and stem cell research

Electrical cell manipulation in microfluidic systems

This dissertation reports on the development of devices and concepts for electrical and microfluidic cell manipulation. In the present context, the term cell manipulation stands for both cell handling and cell modification. The combination of microfluidic channels with micropatterned electrodes allows for the definition of highly localised chemical and electrical environments with spatial resolution comparable to the size of a cell. The devices fabricated in the frame of this thesis employ dielectrophoretic particle handling schemes such as deflection and trapping in pressure-controlled laminar flows to bring cells to – or immobilise them at – locations where cell altering electric fields or chemicals are present. The two concepts of dielectrophoretic cell dipping and cell immersion are introduced and experimentally shown for erythrocytes dipped into Rhodamine in flow, and for individually immobilised Jurkat cells immersed by Trypan Blue. Also, in-situ membrane breakdown in high intensity AC electric fields is optically assessed by efflux of haemoglobin (haemolysis) and by influx of nucleic stains or fluorescence-enhancing ions. The most advanced experiments are on-chip medium exchange followed immediately by electropermeablisation or electrodeformation. The majority of assays presented in this thesis are carried out in microfabricated glass-polymer-glass chips featuring top-bottom electrodes. The devices are fluidically controlled by external gas pressure bridging circuits. Experimental evidence of the unmatched precision of pressure bridging is given in the case of micrometric xy positioning of cells at the intersection of two perpendicular microfluidic channels. Further shown in this document are two methods of optical in-situ temperature measurements, important for bioinstrument characterisation. The two concepts of thermoquenching of a fluorescent dye and the original thermoprecipitation of "smart polymers" are used. The last part of this work deals with the innovative, conceptual engineering tool Liquid Electrode. The general concept and its advantages over solid-state electrodes are given, followed by numerical particle tracking in the case of the novel lateral nDEP particle deflection. The chapter on liquid electrodes concludes with preliminary experimental results of buffer swapping of cells in flow and of AC electropermeabilisation of erythrocytes at frequencies far below the cut-off frequency of corresponding solid-state microelectrodes