Microdevices and Microsystems for Cell Manipulation

Microfabricated devices and systems capable of micromanipulation are well-suited for the manipulation of cells. These technologies are capable of a variety of functions, including cell trapping, cell sorting, cell culturing, and cell surgery, often at single-cell or sub-cellular resolution. These functionalities are achieved through a variety of mechanisms, including mechanical, electrical, magnetic, optical, and thermal forces. The operations that these microdevices and microsystems enable are relevant to many areas of biomedical research, including tissue engineering, cellular therapeutics, drug discovery, and diagnostics. This Special Issue will highlight recent advances in the field of cellular manipulation. Technologies capable of parallel single-cell manipulation are of special interest

Nanopipettes as Monitoring Probes for the Single Living Cell: State of the Art and Future Directions in Molecular Biology.

Examining the behavior of a single cell within its natural environment is valuable for understanding both the biological processes that control the function of cells and how injury or disease lead to pathological change of their function. Single-cell analysis can reveal information regarding the causes of genetic changes, and it can contribute to studies on the molecular basis of cell transformation and proliferation. By contrast, whole tissue biopsies can only yield information on a statistical average of several processes occurring in a population of different cells. Electrowetting within a nanopipette provides a nanobiopsy platform for the extraction of cellular material from single living cells. Additionally, functionalized nanopipette sensing probes can differentiate analytes based on their size, shape or charge density, making the technology uniquely suited to sensing changes in single-cell dynamics. In this review, we highlight the potential of nanopipette technology as a non-destructive analytical tool to monitor single living cells, with particular attention to integration into applications in molecular biology

Estimation of Injection Volume in Capillary Microinjection Using Electrical Impedance Measurement

Capillary pressure microinjection (CPM) is a tool for transporting small sample volumes into living cells utilizing a sharp glass pipette and pressure pulses. The automation level of the current state-of-the-art microinjection devices is low and this makes the technique slow, imprecise and inefficient. The objective of this thesis work is to develop a method to estimate the injection volume in the capillary pressure microinjection technique of living adherent cells. This method would improve the reliability and repeatability of CPM and facilitate automating the injection procedure. Due to the extremely small dimensions involved in the process, a straight measurement of the injection volume is not possible. The strategy used in this work is to generate a mathematical model for the injection volume as a function of the injection pressure and the pipette electrical resistance. A measurement setup is built around a microinjection system to gather data for constructing the model. The injection pressure is measured with a pressure sensor, the pipette electrical resistance is determined using a custom-made impedance measurement circuitry and the injection volume is estimated by using a fluorescent dye as the injection liquid and recording image data from the injections. Several injection pressures and micropipette sizes are used to achieve data extensively enough. A MATLAB based automated algorithm is generated to handle the measurement data and organize the results efficiently. The measurement results give a rough estimate of the relationship between the injection volume, the injection pressure and the pipette electrical resistance. However, a reliable model cannot be built based on the data. The reason is the rather limited amount of suitable measurement data for modelling it was possible to collect due to the numerous error situations. Nevertheless, new important information of the nature of the microinjection procedure is obtained and valuable observations on measurements connected to microinjection are made. Further studies must be done to solve the problems in the tests to be able to gather the data more efficiently and construct the actual model. /Kir1

Integration of single-cell electropermeabilization together with electrochemical measurement of quantal exocytosis on microchips

An electrochemical microelectrode located immediately adjacent to a single neuroendocrine cell can record spikes of amperometric current that result from quantal exocytosis of oxidizable transmitter from individual vesicles. Using electroporation we have developed an efficient method where the same electrochemical microelectrode is used to electropermeabilize an adjacent chromaffin cell and then measure the consequent quantal catecholamine release using amperometry. Trains of voltage pulses, 5-7 V in amplitude and 0.1-0.2 ms in duration can reliably trigger release from cells using gold electrodes. Amperometric spikes induced by electropermeabilization have similar areas, peak heights and durations as amperometric spikes elicited by depolarizing high K+ solutions. Uptake of trypan blue stain into cells demonstrated that the plasma membrane is permeabilized by the voltage stimulus. Robust quantal release is elicited upon electroporation in 0 Ca2+/5 mM EGTA in the bath solution. Electropermeabilization-induced transmitter release requires Cl- in the bath solution--bracketed experiments demonstrate a steep dependence of the rate of electropermeabilization-induced transmitter release on [Cl- ] between 2 and 32 mM. Using the same electrochemical electrode to electroporate and record quantal release of catecholamines from an individual chromaffin cell allows precise timing of the stimulus, stimulation of a single cell at a time, and can be used to load membrane impermeant substances into a cell.Includes bibliographical references (pages 110-128)

SINGLE-CELL ELECTROPORATION USING ELECTROLYTE-FILLED CAPILLARIES WITH MICRO-SCALE TIPS

Single-cell electroporation (SCEP) is a recently developing powerful technique for cell analysis and cell manipulation. In the first chapter of this thesis, a review about the theories and techniques is fulfilled, including a detailed description of the factors affecting SCEP, and a discussion about how to optimize SCEP for high efficiency and survivability. Based on the previous experimental results and numerical simulation, a hypothesis is proposed which leads us to find that small tips could be a solution to electroporate small cells with simultaneous maximization of electroporation efficiency and survivability when using electrolyte-filled capillaries (EFC) with pulled tips.In the second chapter, an integrated circuit for SCEP and controlling is demonstrated. EFCs with 2 &mu tips are constructed and used for SCEP of A549 cells with an extremely high spatial resolution. Distance between tip and cell is revealed to be vital in SCEP because of its direct control of the local electric field distribution and strength; to control distance precisely, a current measurement method inspired by tip-cell giga-seals is applied. High temporal resolution videos hint an abrupt intracellular fluorescence loss at the time scale of pulse duration followed by recovery in the small portion of cell membrane facing the tips. Viability of cells is highly related to the fluorescence loss, fluorescence exposure and dye types. Comsol simulation using the real shape capillaries helps to guide the electroporation throughout our experiments.However, this protocol evokes overcoming technical difficulties in terms of getting high survivability and decreasing variance, which are our two main aims. The advantage of small tips and the hypothesis are still to be examined. This is referred in the third chapter, as well as other following-up future work

Microchannel enhanced neuron-computer interface: design, fabrication, biophysics of signal generation, signal strength optimization, and its applications to ion-channel screening and basic neuroscience research

En el presente trabajo, utilizamos técnicas de microfabricación, simulaciones numéricas, experimentos de electrofisiología para explorar la viabilidad en me- jorar la interface ordenador-neurona a través de microcanales, y la biofísica para la generación de señales en los dispositivos con microcanales. También demos- tramos que los microcanales pueden ser usados como una técnica prometedora con alto rendimiento en el muestreo automático de canales iónicos a nivel subce- lular. Finalmente, se ha diseñado, fabricado y probado el micropozo-microcanal como modificación adicional a los arreglos de multielectrodos, permitiendo una alta ganancia en la relación señal/ ruido (en inglés Signal to Noise Ratio SNR), y el registro de múltiples-lugares en poblaciones de baja densidad de redes neu- ronales del hipocampo in vitro. Primero, demostramos que son de alto rendimiento los microcanales de bajo costo con interface neurona-electrodo, para el registro extracelular de la activi- dad neuronal con baja complexidad, por periodos estables de larga duración y con alta ganancia SNR. En seguida, se realiza un estudio mediante experimentos y simulaciones nu- méricas de la biofísica para la generación de las señales obtenidas de los dispositi- vos con microcanales. Basados en los resultados, racionalizamos y demostramos como es que la longitud del canal (siendo 200 μm) y la sección transversal del microcanal (siendo 12 μm2) canaliza a los potenciales de acción para estar dentro del rango de milivolts. A pesar del bajo grado de complexidad envuelto en la fabricación y aplicación, los dispositivos con microcanales otorgan una sola media de valor SNR de 101 76, lo cual es favorablemente comparable con la SNR que se obtiene de desarrollos recientes que emplean electrodos curados con CNT y Si-NWFETs. Más aún, nosotros demostramos que el microcanal es una técnica promete- dora para el alto rendimiento del muestro automático de canales iónicos a nivel subcelular: (1) Información experimental y simulaciones numéricas sugieren que las señales registradas sólo afectan los parches membranales localizados dentro del microcanal o alrededor de 100 μm de las entradas del microcanal. (2) La transferencia de masa de los componentes químicos en los microcanales fue ana- lizada por experimentos y simulaciones FEM. Los resultados muestran que los microcanales que contienen glía y tejido neuronal pueden funcionar como barre- ra de fluido/química. Los componentes químicos pueden ser solamente aplicados a diferentes compartimentos a nivel subcelular. Finalmente, basado en simulaciones numéricas y resultados experimentales, se propone que del micropozo-microcanal, obtenido de la modificación de MEA (MWMC-MEA), la longitud óptima del canal debe ser 0,3 mm y la posición 1 óptima del electrodo intracanal, hacia la entrada más cercana del microcanal, debe ser 0,1 mm. Nosotros fabricamos un prototipo de MWMC-MEA, cuyo hoyo pasante sobre las películas de Polydimethylsiloxane (PDMS) fue microtrabajado a través de la técnica de grabados reactivos de plasma de iones. La baja densidad del cultivo (57 neuronas /mm2) en el MWMC-MEAs permitió que las neuronas vivieran al menos 14 días, con lo que la señal neuronal con la máxima SNR obtenida fue de 142. 2In this present work, we used microfabrication techniques, numerical simulations, electrophysiological experiments to explore the feasibility of enhancing neuron-computer interfaces with microchannels and the biophysics of the signal generation in microchannel devices. We also demonstrate the microchannel can be used as a promising technique for high-throughput automatic ion-channel screening at subcellular level. Finally, a microwell-microchannel enhanced multielectrode array allowing high signal-to-noise ratio (SNR), multi-site recording from the low-density hippocampal neural network in vitro was designed, fabricated and tested. First, we demonstrate using microchannels as a low-cost neuron-electrode interface to support low-complexity, long-term-stable, high SNR extracellular recording of neural activity, with high-throughput potential. Next, the biophysics of the signal generation of microchannel devices was studied by experiments and numerical simulations. Based on the results, we demonstrate and rationalize how channels with a length of 200 μm and channel cross section of 12 μm2 yielded spike sizes in the millivolt range. Despite the low degree of complexity involved in their fabrication and use, microchannel devices provided a single-unit mean SNR of 101 76, which compares favourably with the SNR obtained from recent developments employing CNT-coated electrodes and Si-NWFETs. Moreover, we further demonstrate that the microchannel is a promising technique for high-throughput automatic ion-channel screening at subcellular level: (1) Experimental data and numerical simulations suggest that the recorded signals are only affected by the membrane patches located inside the microchannel or within 100 μm to the microchannel entrances. (2) The mass transfer of chemical compounds in microchannels was analyzed by experiments and FEM simulations. The results show that the microchannel threaded by glial and neural tissue can function as fluid/chemical barrier. Thus chemical compounds can be applied to different subcellular compartments exclusively. Finally, a microwell-microchannel enhanced MEA (MWMC-MEA), with the optimal channel length of 0.3 mm and the optimal intrachannel electrode position of 0.1 mm to the nearest channel entrance, was proposed based on numerical simulation and experiment results. We fabricated a prototype of the MWMCMEA, whose through-hole feature of Polydimethylsiloxane film (PDMS) was micromachined by reactive-ion etching. The low-density culture (57 neurons/mm2) were survived on the MWMC-MEAs for at least 14 days, from which the neuronal signal with the maximum SNR of 142 was obtained

Manipulating Cardiovascular Cellular Interactions and Mechanics: A Multidimensional and Multimodal Approach

The goal of this dissertation is to better understand cellular mechanics across length scales for the development of computational models of tissue behavior. To this end, we had two major approaches, multidimensional and multimodal. Firstly, to use a model that better mimics in vivo like cellular environment, microtissue (spheroid) cell culture system was used to study cell mechanics. Secondly, a novel technique was designed to study single cell mechanics in multiple dimensions. Cell mechanical properties are directly related to the composition and organization of the cytoskeleton, which is physically coupled to neighboring cells through adherens junctions and to extracellular matrix through focal adhesion complexes. As such, we hypothesize that the variations in cellular interactions affects cell mechanics. To test our hypothesis, cardiomyocytes and vascular smooth muscle microtissues were cultured under several conditions that limited the cell-cell and cell-matrix interactions. Cell interactions facilitated by integrin β1, connexin 43, and N-cadherin was inhibited and their effect on cell stiffness was characterized by atomic force microscopy (AFM). Currently, there does not exist a single technique that can measure mechanics of a single cell in two different dimensions. To address this gap, we designed a novel set up that combines two different single cell mechanics measurement techniques, AFM and carbon fiber. This combination allows for characterization of mechanical properties of single cells in multiple dimensions. The results of these studies provide insights from a basic science perspective. The results provide information regarding cell mechanics in multiple dimensions at both single cell as well microtissue level. The ultimate fulfillment of this work would be its incorporation into a multiscale model, leading to the ability to tie macro- scale behaviors to nano- scale phenomenon. Such models may help to better understand tissue behavior and further our understanding of the etiology of many diseases

Atomic force microscopy-based mechanobiology

Mechanobiology emerges at the crossroads of medicine, biology, biophysics and engineering and describes how the responses of proteins, cells, tissues and organs to mechanical cues contribute to development, differentiation, physiology and disease. The grand challenge in mechanobiology is to quantify how biological systems sense, transduce, respond and apply mechanical signals. Over the past three decades, atomic force microscopy (AFM) has emerged as a key platform enabling the simultaneous morphological and mechanical characterization of living biological systems. In this Review, we survey the basic principles, advantages and limitations of the most common AFM modalities used to map the dynamic mechanical properties of complex biological samples to their morphology. We discuss how mechanical properties can be directly linked to function, which has remained a poorly addressed issue. We outline the potential of combining AFM with complementary techniques, including optical microscopy and spectroscopy of mechanosensitive fluorescent constructs, super-resolution microscopy, the patch clamp technique and the use of microstructured and fluidic devices to characterize the 3D distribution of mechanical responses within biological systems and to track their morphology and functional state.Peer ReviewedPostprint (published version

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

A modular multi electrode array system for electrogenic cell characterisation and cardiotoxicity applications

Multi electrode array (MEA) systems have evolved from custom-made experimental tools, exploited for neural research, into commercially available systems that are used throughout non-invasive electrophysiological study. MEA systems are used in conjunction with cells and tissues from a number of differing organisms (e.g. mice, monkeys, chickens, plants). The development of MEA systems has been incremental over the past 30 years due to constantly changing specific bioscientific requirements in research. As the application of MEA systems continues to diversify contemporary commercial systems are requiring increased levels of sophistication and greater throughput capabilities. [Continues.