Dielectrophoretic discrimination of pluripotent myoblast with Raman spectroscopic analysis of the cell plasma membrane for application in Huntington's disease

Myoblasts are muscle derived mesenchymal stem cell progenitors that have great potential for use in regenerative medicine, especially for cardiomyogenesis grafts and intracardiac cell transplantation. To utilise such cells for pre -clinical and clinical applications, and especially for personalized medicine, it is essential to generate a synchronised, homogenous, population of cells that display phenotypic and genotypic homogeneity within a population of cells. This thesis demonstrates that the biomarker -free technique of dielectrophoresis (DEP) can be used to discriminate cells between stages of differentiation in the C2C12 myoblast pluripotent mouse model. Terminally differentiated myotubes were separated from C2C12 myoblasts to better than 96% purity, a result validated by flow cytometry and Western blotting. To determine the extent to which cell membrane capacitance, rather than cell size, determined the DEP response of a cell, C2C12 myoblasts were co- cultured with GFP- expressing fibroblasts of comparable size distributions (mean diameter -10 gm). A DEP sorting efficiency greater than 98% was achieved for these two cell types, a result concluded to arise from the fibroblasts possessing a larger membrane capacitance than the myoblasts. It is currently assumed that differences in membrane capacitance primarily reflect differences in the extent of folding or surface features of the membrane. However, our finding by Raman spectroscopy that the fibroblast membranes contained a smaller proportion of saturated lipids than those of the myoblasts suggests that the membrane chemistry should also be taken into account.These high levels of discrimination raised more questions about the cell plasma membrane characteristics that may be responsible for the dielectrophoretic response. This prompted to extend the work to a specific neurodegenerative disease, Huntington's disease. Several studies have been revealing the association between plasma membrane dysregulation and Huntington's disease. In particular the feasibility to use peripheral fibroblasts cells from donors affected by the disease, as a forecasting model marker for Huntington. Although there are substantial evidences about the indirect effect of the disease on the plasma membrane, a non -invasive technique that can discriminate and characterise a cell sample is not available. Raman spectroscopy with associated statistical multivariate analysis was used to characterise sub -cellular differences in extracted plasma membranes from peripheral fibroblastic cells in order to elucidate the differences between cells affect and non - affected by the disease. The results clearly showed that indeed the plasma membrane carries differences that can be attributed to the presence of the disease making the plasma membrane an amenable and novel biomarker for Huntington's diseas

Single-cell microfluidic impedance cytometry: From raw signals to cell phenotypes using data analytics

The biophysical analysis of single-cells by microfluidic impedance cytometry is emerging as a label-free and high-throughput means to stratify the heterogeneity of cellular systems based on their electrophysiology. Emerging applications range from fundamental life-science and drug assessment research to point-of-care diagnostics and precision medicine. Recently, novel chip designs and data analytic strategies are laying the foundation for multiparametric cell characterization and subpopulation distinction, which are essential to understand biological function, follow disease progression and monitor cell behaviour in microsystems. In this tutorial review, we present a comparative survey of the approaches to elucidate cellular and subcellular features from impedance cytometry data, covering the related subjects of device design, data analytics (i.e., signal processing, dielectric modelling, population clustering), and phenotyping applications. We give special emphasis to the exciting recent developments of the technique (timeframe 2017-2020) and provide our perspective on future challenges and directions. Its synergistic application with microfluidic separation, sensor science and machine learning can form an essential tool-kit for label-free quantification and isolation of subpopulations to stratify heterogeneous biosystems

Biological response of Chlorella vulgaris to pulsed electric field treatment for improvement of protein extraction

Angesichts des Klimawandels und einer stetig wachsenden Weltbevölkerung können Mikroalgen eine wichtige Rolle als nachhaltige Energie- und Nahrungsquelle der Zukunft spielen. Zur Extraktion wertvoller Inhalts- und Nährstoffe ist ein Zellaufschluss notwendig. Die Elektroimpulsbehandlung (EIB) bietet eine energieeffiziente und schonende Alternative im Vergleich zu mechanischen Zellaufschlussmethoden. Jedoch sind die biologischen Prozesse und zellulären Mechanismen hinter dem Zelltod nach EIB noch wenig untersucht. Aus diesem Grund wurden die einzellige grüne Mikroalge Chlorella vulgaris und das Cyanobakterium Spirulina als Modellorganismen verwendet, um die Wirkung von EIB auf biologische Zellen zu untersuchen. Dafür wurde eine Methode zur Überwachung der Viabilität nach EIB unter Verwendung von Fluoresceindiacetat (FDA) in C. vulgaris etabliert. Im Anschluss wurden die experimentellen EIB-Parameter so eingestellt, dass ein fixes Verhältnis von Zellen nach der Behandlung abstirbt, während der andere Teil überlebt. Mit diesen Werkzeugen war eine quantitative Analyse des Zelltodes nach EIB möglich. Basierend auf den Analyseergebnissen wurde die EIB-Extraktion von Proteinen und dem wertvollen blauen Farbstoff Phycocyanin aus Spirulina unter verschiedenen post-EIB Inkubationsbedingungen untersucht. Zur Optimierung der Elektroextraktionseffizienz in Spirulina wurden die Einflüsse des pH des externen Mediums, der Biomassekonzentration, der Zellaggregation sowie der Energiereduktion untersucht. Das optimierte Elektroextraktionsprotokoll mit höherer Biomassekonzentration und geringerer Behandlungsenergie erfordert eine post-EIB-Inkubation unter kontrollierten Bedingungen (Raumtemperatur, pH 6 oder 8, homogene Suspension), die für die Freisetzung und Stabilität von Phycocyanin entscheidend sind. Mit diesem Wissen besteht eine mögliche biotechnologische Anwendung darin, schonende EIB mit niedrigstem Energieeintrag durchzuführen, was zu einer effizienten Protein- und Phycocyanin-Gewinnung führt. An C. vulgaris konnte gezeigt werden, dass EIB mit niedrigem Energieeintrag auch als abiotisches Stresssignal wirken kann. Dies wurde sichtbar in Form einer gestörten Redox-Homöostase, bei der sowohl die Freisetzung von Wasserstoffperoxid als auch Lipidoxidation gemessen werden konnten. Die Hemmung von Prozessen, die mit dem programmierten Zelltod (PCD) zusammenhängen, zeigten, dass höchstwahrscheinlich Ca-Signalwege, Aktindynamik und Membranversteifung keine notwendige Rolle beim EIB-induzierten Zelltod spielen. Die Freisetzung von Cytochrom f konnte nur im Hochdruckhomogenisations (HPH) Extrakt und nicht nach EIB nachgewiesen werden. Zellsuspensionen mit hoher Zelldichte, die an der Überlebensschwelle gepulst wurden, zeigten nur eine langsame Manifestation des Zelltods. Dies führte zur Entdeckung eines Zelltod-induzierenden Faktors (CDIF). Es konnte nachgewiesen werden, dass durch EIB und HPH-Behandlung der CDIF aus C. vulgaris extrahiert werden kann. Wasserlöslicher Extrakt, der diesen CDIF enthielt, führte zum Absterben von unbehandelten Mikroalgen (insbesondere nur bei C. vulgaris). Weitere Experimente zeigten die Entstehung des CDIF in der stationären Wachstumsphase, Hitzelabilität und Dosisabhängigkeit. Ebenso wie die Empfindlichkeit gegenüber direkter EIB hing die Empfindlichkeit der Empfängerzellen gegenüber dem CDIF vom Zellzyklusstadium ab. Untersuchungen zur Extraktionseffizienz von Proteinen aus C. vulgaris führten zu dem Ergebnis, dass die erforderliche spezifische Energie für maximalen Ertrag der zuvor bestimmten Behandlungsenergie an der Überlebensschwelle entspricht. Alle experimentellen Ergebnisse weisen darauf hin, dass der EIB-induzierte Zelltod und die damit verbundene hohe Extraktionseffizienz nicht nur auf rein physikalische Phänomene zurückzuführen sind, sondern einen biologischen Prozess beinhalten müssen. Das Arbeitsmodell bezüglich des CDIF beinhaltet, dass der Faktor aus zellwandabbauenden Enzymen wie Chitinasen besteht. EIB bei sehr geringem Energieeintrag wirkt als abiotisches Stresssignal. In Kombination mit einer beschädigten Zellintegrität aufgrund von Poren in der Zellmembran führen PCD-Prozesse zu einer enzymatischen Autolyse, bei der der CDIF (Chitinasen) freigesetzt wird. Die Zellwand wird durch den CDIF geschwächt. Wird der CDIF-haltige Extrakt unbehandelten Empfängerzellen zugesetzt, zeigt er zunächst über den Zellwandabbau eine äußere Wirkung. Nach Internalisierung kann der CDIF als internes Signal fungieren, das PCD auslöst

DEVELOPMENT OF A XENOGRAFT FOR ANNULAR REPAIR USING PULSED ELECTRIC FIELD EXPOSURES FOR ENHANCED DECELLULARIZATION

Severe back injuries and chronic pain necessitate surgical replacement of damaged intervertebral disc (IVD) cartilage in advanced disease stages. Bovine IVD tissue has been exposed to an upper threshold pulsed electric field (PEF) dose, causing cell death without thermal damage to the tissue. Subsequent PEF exposures at lower magnitudes have accelerated the removal of immunogenic biomolecules though electrokinetic extraction using optimized aqueous solutions. This approach yields a natural scaffold, ready for biocompatibility and mechanical strength assessment. The effects of microsecond pulsed electric fields (µsPEF) on primary bovine AF fibroblast-like cells have been characterized in vitro. PEFs of 10 and 100 µs durations, with varying numbers of pulses and electric field strengths, were applied to the cells. Furthermore, a low-intensity and minimally heating PEF-induced electrokinetic flow for molecular extraction has been established, involving the determination of the electrophoretic mobility of charged molecules within the AF cartilage tissue. Ultimately, a biocompatible AF scaffold has been generated using PEF and electrolyte solutions in a custom-designed bioreactor. Compared to chemical methods that take days, PEF application achieves decellularization of cartilage tissue within three hours, while preserving the desired biomolecules and ultrastructure of the tissue matrix

Label-free and Multi-parametric Monitoring of Cell-based Assays with Substrate-embedded Sensors

Various approaches have been pursued on the basis of electrochemical or piezoelectric transducers, particularly of the quartz crystal microbalance (QCM), to monitor non-invasively and in real-time cellular states and reactions with substrate-embedded sensors. On the one hand, these comprised the technical development of piezoelectric sensors with multiple read-out spots and the integration of additional non-invasive (electro- and optochemical) sensor technologies on the QCM surface. On the other hand, a variety of studies and cell-based assays (CBAs) have been performed in order to test the sensor performances and to gain a deeper understanding of the sensors’ readout parameters with respect to their information content about the biophysical properties and the metabolic behavior of cells. Fig. 7–1 presents an overview of the different projects on the basis of QCM sensor disks presented in this thesis. In the first project (Fig. 7–1 A) a novel electrode layout was designed on the basis of commercial 5 MHz AT-cut quartz disks to implement two independent readout spots on the QCM surface. This also comprised the construction of new measurement chambers for the electrical actuation and sensing of quartz oscillations. These two-electrode QCM sensors (2ElQ) are also referred to as multichannel QCM (MQCM). The developed MQCM sensor spots on one quartz disk exhibited a strong interference, even though they were operated sequentially, which is in contrast to the results of previous theoretical calculations. The resonances could be successfully decoupled by coating half of the quartz surface and one sensor spot, respectively, with a thin and rigid film of photoresist. This quartz loading with mass caused a shift in the resonance spectra of the coated resonator to lower frequencies and efficient decoupling. The operation of the decoupled MQCM sensors demonstrated both, a sensitive and equal change in the oscillation characteristics of the two resonators upon loading of the quartz with medium. The Q-factor was not significantly different for the two resonators, qualifying the MQCM for its application in CBAs. Building on the preceding development of the double-electrode quartz disks, a novel electrode layout has been realized at the sensor surface, which enables the complementary electrochemical (impedance spectroscopical) characterization of the substrate-liquid interface in addition to its mechanical characterization by the piezoelectric transducers (Fig. 7–1 B). This layout was achieved by removing a small area of the insulating photoresist on the coated electrode in the photolithographic process. By this, a coplanar electrode arrangement of a small working electrode and a bigger counter electrode was created. This sensor combination on the basis of the MQCM is an improvement of the so-called 2nd generation QCM to what we call the 3rd generation QCM, and which is also referred to as QCM-ECIS. Various electrode layouts, varying in size and number of the working electrode(s), were fundamentally characterized microscopically and by profilometry regarding the geometrical properties and by means of impedance spectroscopy with respect to the sensing performances in QCM- and ECIS-mode. An optimal electrode layout was identified and defined as standard for subsequent applications in CBAs. In both QCM- and ECIS studies of cell-covered sensor surfaces significant changes in the characteristic sensing parameters with respect to the cell-free electrodes are measurable. In addition to the measurement of absolute signal changes, the transducer technologies of QCM and ECIS also enable to monitor the kinetic changes of the readout parameters with high temporal resolution. This allows to use the dual sensors for monitoring and analyzing the states of adherent cell cultures in any kind of assay, label-free, non-invasively, and in real-time. Mechanical (QCM-mode) and the dielectric (ECIS-mode) characteristics of cell adhesion were simultaneously measured for two different cell lines (MDCK II and NRK), with high reproducibility for each. The total and kinetic parameter changes in both sensing modes distinguished clearly and were specific for the cell lines under test. The signals from both QCM-mode and ECIS-mode recordings also reported on significant impacts of the presence/ absence of bivalent cations (Ca2+, Mg2+) on the attachment and spreading kinetics and behavior of MDCK II cells. Aside from cell adhesion studies, the cytomechanical and cell morphological reactions towards various stimuli were monitored and analyzed by QCM-ECIS in a multitude of cellular assays: systematic softening and stiffening of cells (using agents for disassembling the actin cytoskeleton and cross-linking protein structures, respectively), intracellular stimulation (using a second messenger analogue), as well as electrical manipulation (electroporation (ELPO) and wounding) of cell layers (applying invasive voltage pulses). The applicability of electrical actuation and the subsequent non-invasive, time-resolved, and dual sensing with the electrodes of the QCM-ECIS substrates has been successfully demonstrated. The monitoring of CBAs with the dual QCM-ECIS sensor chips developed in this thesis provides not only a multiplication of the information gain due to the complementarity of QCM and ECIS readout parameters. The simultaneous, time-resolved measurements also enable the kinetic correlation of the sensor signals in novel 2 D and 3 D diagrams, which offers the hitherto unprecedented opportunity for a more detailed view and analysis of the coherence or consecutiveness of mechanical and morphological/ dielectric changes of a cell layer under study. A third research project focused on the combination of optical-chemical sensors (OCS) with the piezoelectric (QCM) sensor technology. For this purpose, the quartz crystal surface was coated with a polymer film with embedded phosphorescent indicator dye for the target analyte. The luminescence properties were measured by means of fluorescence (phosphorescence) lifetime imaging (FLIM). By using a temperature-sensitive paint (TSP), an increase in temperature on the sensor surface upon high-amplitude oscillations was monitored and imaged this way in one project (Fig. 7–1 C). Based on this experimentally determined local heating on the QCM surface and the thereby generated temperature gradient in the liquid above the resonator, a thermophoretic convection in the fluid has been simulated. Theoretical considerations showed that the convection profile in the measurement vessel counteracts and even largely prevents the sedimentation of cells onto the sensor surface. It is suggested that the effect of thermophoresis is crucial especially in studies of biomolecular interactions on QCM surfaces at elevated shear amplitudes and driving voltages, respectively, which however has not been considered in literature to date. The phosphorescence quenching capability of oxygen was utilized in a second imaging project to monitor and image the local concentration and distribution of oxygen on the growth substrate of cells by means of a so-called pressure(/oxygen)-sensitive paint (PSP) (Fig. 7–1 D). A home-made experimental setup was constructed for sensor calibration and the imaging of subcellular oxygen, consisting of a FLIM setup coupled to an upright microscope and a temperature- and oxygen-controlled calibration and measurement chamber suitable for cellular applications. The cytocompatible sensor films have been characterized under various test conditions (in air, under medium, at different temperatures) regarding their sensitivity and response characteristics to different oxygen partial pressures. The oxygen consumption of cells adherently grown on the sensor film was successfully monitored and imaged by this setup. The time-resolved measurements demonstrated a significantly faster consumption of oxygen of a cell layer stimulated with a respiration chain decoupler compared to an unstimulated control cell layer. Taken together, various technical improvements of piezoelectric sensors (QCM) have been realized (MQCM, QCM-ECIS, ELPO-QCM-ECIS, QCM-OCS), which provide a significant information gain in cell-based applications. The sensors developed enable the high-content screening (HCS) of adherent cell lines in a wide range of assay formats and provide complementary physico-chemical information for obtaining a more complete picture of the state of cells and their reactions in contact to diverse stimuli. All sensor techniques share the characteristics of time-resolved, label-free, and non-invasive monitoring. This allows to disclose and analyze even the kinetics, delayed effects, recoveries, and fluctuations of physicochemical alterations of a studied cell layer, in addition to the absolute parameter changes, which is a valuable improvement compared to classical endpoint assays. The approach of combined, independent sensor systems also provides the novel possibility to bring parameters obtained by the different readout technologies from one cell layer in a temporal correlation, by which new insights into physiological relationships are possible

Cellular Analysis by Atomic Force Microscopy

Exocytosis is a fundamental cellular process where membrane-bound secretory granules from within the cell fuse with the plasma membrane to form fusion pore openings through which they expel their contents. This mechanism occurs constitutively in all eukaryotic cells and is responsible for the regulation of numerous bodily functions. Despite intensive study on exocytosis the fusion pore is poorly understood. In this research micro-fabrication techniques were integrated with biology to facilitate the study of fusion pores from cells in the anterior pituitary using the atomic force microscope (AFM). In one method cells were chemically fixed to reveal a diverse range of pore morphologies, which were characterised according to generic descriptions and compared to those in literature. The various pore topographies potentially illustrates different fusion mechanisms or artifacts caused from the impact of chemicals and solvents in distorting dynamic cellular events. Studies were performed to investigate changes in fusion pores in response to stimuli along with techniques designed to image membrane topography with nanometre resolution. To circumvent some deficiencies in traditional chemical fixation methodologies, a Bioimprint replication process was designed to create molecular imprints of cells using imprinting and soft moulding techniques with photo and thermal activated elastomers. Motivation for the transfer of cellular ultrastructure was to enable the non-destructive analysis of cells using the AFM while avoiding the need for chemical fixation. Cell replicas produced accurate images of membrane topology and contained certain fusion pore types similar to those in chemically fixed cells. However, replicas were often dehydrated and overall experiments testing stimuli responses were inconclusive. In a preliminary investigation, a soft replication moulding technique using a PDMS-elastomer was tested on human endometrial cancer cells with the aim of highlighting malignant mutations. Finally, a Biochip comprised of a series of interdigitated microelectrodes was used to position single-cells within an array of cavities using positive and negative dielectrophoresis (DEP). Selective sites either between or on the electrode were exposed as cavities designed to trap and incubate pituitary and cancer cells for analysis by atomic force microscopy (AFMy). Results achieved trapping of pituitary and cancer cells within cavities and demonstrated that positive DEP could be used as a force to effectively position living cells. AFM images of replicas created from cells trapped within cavities illustrated the advantage of integrating the Biochip with Bioimprint for cellular analysis

Interfacial Interactions between Implant Electrode and Biological Environment

Electrodes implanted into neural systems are known to degrade due to encapsulation by surrounding tissues. The mechanisms of electrode-tissue interactions and prediction of the behavior of electrode are yet to be achieved. This research will aim at establishing the fundamental knowledge of interfacial interactions between the host biological environment and an implanted electrode. We will identify the dynamic mechanisms of such interfacial interactions. Quantitative analysis of the electrical properties of interface will be conducted using Electrochemical Impedance Spectroscopy (EIS). Results will be used to develop a general model to interpret electrical circuitry of the interface. This is expected to expand our understanding in the effects of interfacial interactions to the charge transport. The interfacial interactions of an implanted electrode with neural system will be studied in two types of electrodes: silver and graphene coated. The interfacial impedance of both samples will be studied using EIS. The development of the cellular interaction will be investigated using histological procedure. X-ray photoemission spectroscopy (XPS) will be employed to study the chemical effects on the silver electrodes. Atomic force microscopy and Raman spectroscopy will be used for material characterization of graphene-coated electrodes. In the study of silver electrode, two mechanisms affecting the interfacial impedance are proposed. First is the formation of silver oxide. The other is the immuno-response of tissue encapsulation. Histological results suggest that higher cell density cause higher impedance magnitude at the interface. It is also found that the cellular encapsulation dominates the increase in impedance for longer implanted time. In the study of graphene-coated electrode, it is found that the graphene can strongly prevent the metal substrate from being oxidized. It not only provides good electrical conductivity for signal transport, but also reduces the speed of the accumulation of tissue around the electrode. Such characteristics of graphene have great potential in the application of neural implant. Finally, the dynamic mechanisms of biological interaction are proposed. A model is also developed to represent the general circuitry of the interface between an implanted electrode and the neural system. The model has three major components, which are interfacial double layer, cellular encapsulation, and the substrate. The model presented in this study can compensate for selection and prediction of materials and their behaviors

Impedance spectroscopy for in vitro toxicology

The impedance of biological material changes with frequency, a phenomenon that has been discovered more than 100 years ago. It is due to the fact that the cell membrane acts as a capacitor which filters out currents at low frequency and lets them pass at high frequency. This fundamental knowledge about biological dielectrics has incompletely been exploited to detect and distinguish toxicity effects on cell cultures, although impedance measurements have been used for long in this field. In this thesis, it was found that low frequency impedance signals are linked to initial stress responses of cells within cell populations when exposed to a toxin whereas high frequency measurements inform about major cell damage as is indicated by intracellular conductivity changes. In addition, when cells gain resistance to a toxin, they experience a higher cell stiffness which is expressed by an increased low frequency impedance. The study of impedance changes as a function of frequency and drug concentrations lead to the creation of an impedimetric concentration-response map which distinguishes cell responses within four concentration ranges without the use of any label. Although being inherently non-specific, this measurement method was shown to report on distinct toxicity effects, an important prerequisite when studying drug action on cancer cells where stimulating and lethal effects need to be distinguished rigorously. This thesis further encompasses the subject of three-dimensional impedance measurements, i.e. the screening of the entire depth of a three-dimensional tissue culture. Given the success of impedance measurements on cell monolayers, one would expect this development to continue with 3D cultures since the complex structure of in vivo tissues is mimicked more closely and, above all, since rapid and inexpensive techniques which are able to probe thick tissue samples are currently inexistent. Nevertheless, few studies have been carried out in this field. Here, the requirements of three-dimensional impedance sensors are discussed and challenged by the fabrication of a corresponding device, involving the development of so-called gel electrodes through a novel 2-step-soft-lithography process. Their specific design allows for the decrease of leak currents, a common problem when performing three-dimensional impedance measurements. The simultaneous measurement of multiple samples in parallel is an an essential condition when performing high throughput drug toxicity screening. Electrode switch systems are necessary which ultimately lead to setup complexity and signal noises. In this thesis, a method is introduced, enabling the simultaneous implementation of impedance measurements of multiple tissue samples with one electrode pair only. This is simply achieved by exploiting the frequency domain and finally contributed to reducing setup complexity