Rapid determination of general cell status, cell viability, and optimal harvest time in eukaryotic cell cultures by impedance flow cytometry

The determination of cell viability is essential to many areas of life sciences and biotechnology. Typically, cell viability measurements are based on the optical analysis of stained cells, which requires additional labeling steps and is hard to implement online. Frequency-dependent impedance flow cytometry (IFC) provides a label-free, fast, and reliable alternative to determine cell viability at the single cell level based on the Coulter principle. Here, we describe the application of IFC to eukaryotic cell cultures and compare the results to commonly used staining methods. Yeast cell parameters were assessed in normal and heat-inactivated cells as well as in alcoholic fermentation and long-term batch cultures providing a precise and fast determination of the cell viability and further quantitative measures of the cell culture status. As an important new application, we have investigated recombinant protein production in the widely used baculovirus insect cell expression system. The IFC analysis revealed the presence of a subpopulation of cells, which correlates with the protein expression yield, but it is not detectable with conventional optical cell counters. We tentatively identify this subpopulation as cells in the late phase of infection. Their detection can serve as a predictor for the optimal time point of harvest. The IFC technique should be generally applicable to many eukaryotic cell cultures in suspension, possibly also implemented online

Quantitative Macro- and Microscale Methods for Characterizing Cell Viability

The goal of this study is to combine molecular and microdevice methods to characterize and quantify viability of single mammalian cells. Fluorescent-based assays were optimized for adherent HeLa and suspension Jurkat cells and were used as a tool for validation of a microfabricated diagnostic device. Cell and substrate/surface interactions were considered for designing a microfluidic device that can be used to characterize cell viability for quantitative biomedical and cell biology applications, which require label-free, real-time monitoring of cells. Several interdisciplinary methods are employed to evaluate electrical impedance differences between live and dead Jurkat cells in a microfluidic device. Biological Micro-Electro-Mechanical Systems (BioMEMS) offer many advantages over the conventional macroscale approaches to biomedical diagnostics, such as reduced reagents, costs, and power consumption; shorter reaction time; portability; versatility; and potential for parallel, integrated operations, thus having the potential to revolutionize how many current cell-based biomolecular assays are performed. A microchip device to detect cell viability at the single-cell level in real-time has much potential for pharmacological drug screening or point-of-care diagnostics. Optimal cell media conditions such as pH and osmolarity are evaluated to ensure cell viability and adequate sensitivity for detecting cell events via electrical impedance measurements. A fluorescent cell assay using Calcein was optimized for optical validation of Jurkat cell viability studies for cells flowing through a microchannel. Fluorescence microscopy was combined with acquired electrical impedance (at 2 MHz) to validate the presence and viability of each cell at the detection electrodes. The microchip design parameters such as substrate material and geometry of microchannel and electrodes were based of the average 12 um-diameter of Jurkat cells tested. Here, we demonstrate the design of a polymer-based chip device that is able to differentiate between live and dead Jurkat cells on the basis of electrical impedance magnitude and phase signals, which could be related to inherent dielectric differences of live and dead cells. The overall outcome of this study provides groundwork for quantifying cell viability of single cells on-chip, in real-time, in a flow-through system, without the use of expensive fluorescent labels

Strategies to overcome interferences during biomass monitoring with dielectric spectroscopy

Dielectric spectroscopy is extensively used to measure the level of viable biomass during fermentations but can suffer from interference by a variety of factors including the presence of dead cells, bubbles, electric and magnetic fields, changes in the medium composition, conductivity changes and the presence of non-cellular particles. Three different approaches were used to overcome these problems. The first involved the separate measurement of the spectra of the interferent and the cells. If the spectra were significantly different then spectra containing the signals of both cells and the interferent could be deconvoluted to separately determine the relative contribution of the cells and the interferent to the spectra. This deconvolution approach was successfully used to estimate the biomass levels of yeast in the presence of spent grains of barley and hardwood in the medium. A similar approach allowed the interference of electrode polarisation on spectra of yeast and microalgae to be compensated for. An attempt to determine the concentration of non-viable cells in a mixture of dead and live cells was less successful because the signal of the non-viable cells was quite small compared to that of viable cells. A second approach involved the use of a filter to keep the interferent away from the probe surface. This was used successfully in the measurement of the yeast concentration in the presence of spent barley grains. A third approach involved the use of a second sensor in addition to the biomass sensor. This allows the signal of the biomass sensor to be compensated for the interferent. In one set of experiments microelectrodes were developed which were able to confine the electric field to a small volume near the electrode surface. Covering the electrode surface with a gel or a membrane stopped cells from entering this volume whilst allowing medium to diffuse through. This allowed the measurement of changes in the electrical properties of the medium without a contribution by the cells. Whilst this approach worked, the response time was too long for practical use. More successful was the simultaneous measurement of the biomass with an infrared optical probe and a dielectric probe. It was found that the signal of the optical probe was independent of the cell viability, whilst the dielectric probe was quite insensitive to non-viable cells. The combined use of the dielectric probe and the optical probe allowed the culture viability to be determined in a straightforward manner

Nanotechnology enabled microfluidics/Raman spectroscopy systems for bio applications

The vision for this PhD research project was born out of a desire to study the in situ behaviour of suspended nano-materials; specifically, implementing a Raman microscopy system for investigating suspended materials in the microfluidic environment. The author developed a set of innovative research goals to achieve this vision, which include: (1) forming a suitable microfluidic system which can apply controlled forces onto the suspended materials on demand, (2) implementing Raman microscopy to study the behaviour of particles under the influence of such forces while inside the microfluidic system and (3) incorporating the developed microfluidic system for investigating suspended materials of low concentration, including biological cells and surface-enhanced Raman scattering studies. The author implemented the research in three distinct stages such that the work in earlier stages could provide the platform for the future work. In the first stage, the author designed a microfluidic dielectrophoresis platform consisting of curved microelectrodes. This platform was integrated with a Raman microscopy system for creating a novel system capable of detecting suspended particles of various types and spatial concentrations. The system was benchmarked using polystyrene and tungsten trioxide suspended particles, and the outcomes of this novel integrated system showed its strong potential for the determination of suspended particles types and their direct mapping, with several unique advantages over conventional optical systems. In the second stage of this research, the author developed a novel microfluidic-DEP system that could manipulate suspended silver nanoparticles’ spacing in three dimensions. Silver nanoparticles are capable of producing strong surface enhanced Raman scattering (SERS) signals, allowing the Raman system to detect very low concentrations of suspended analytes. DEP provided facile control of the positions and spacings of the suspended silver nanoparticles, and allowed for the creation of SERS hot-spots. The system was studied to determine the optimum DEP and microfluidic flow parameters for generating SERS, and the author was able to demonstrate this as a reversible process. This stage of the research used dipicolinic acid as the target analyte, and the system was demonstrated to have detection limits as small as ~1 ppm concentration levels. In the third stage, the microfluidic-DEP platform was used for trapping and isolating yeast cells. Silver nanoparticles were again used for SERS applications. The trapped cells were interrogated by the Raman system in order to obtain deeper understandings of cells functionalities and their communications under various physical conditions: live vs. dead and isolated vs. grouped. Live vs. dead experiments were conducted as a benchmark, to observe whether SERS is capable of differentiating cells based on the life condition. The research was expanded to study cells that were isolated from one another, and compared those Raman signatures to those from cells in grouped clusters. The author was able to extract unique information from such studies, including the importance of glycine, or proteins with glycine subunits, in the proliferation of yeast cells. The developed system showed great potential as a universal platform for the in situ study of cells, their communications and functionalities

Dielectrophoretic characterization of particles and erythrocytes

Medical lab work, such as blood testing, will one day be near instantaneous and inexpensive via capabilities enabled by the fast growing world of microtechnology. In this research study, sorting and separation of different ABO blood types have been investigated by applying alternating and direct electric fields using class=SpellE\u3edielectrophoresis in microdevices. Poly(dimethylsiloxane) (PDMS) microdevices, fabricated by standard photolithography techniques have been used. Embedded perpendicular platinum (Pt) electrodes to generate forces in AC dielectrophoresis were used to successfully distinguish positive ABO blood types, with O+ distinguishable from other blood types at \u3e95% confidence. This is an important foundation for exploring DC dielectrophoretic sorting of blood types. The expansion of red blood cell sorting employing direct current insulative class=SpellE\u3edielectrophoresis (DC-iDEP) is novel. Here Pt electrodes were remotely situated in the inlet and outlet ports of the microdevice and an insulating obstacle generates the required dielectrophoretic force. The presence of ABO antigens on the red blood cell were found to affect the class=SpellE\u3edielectrophoretic deflection around the insulating obstacle thus sorting cells by type. To optimize the placement of insulating obstacle in the microchannel, COMSOL Multiphysics® simulations were performed. Microdevice dimensions were optimized by evaluating the behaviors of fluorescent polystyrene particles of three different sizes roughly corresponding to the three main components of blood: platelets (2-4 µm), erythrocytes (6-8 µm) and leukocytes (10-15 µm). This work provided the operating conditions for successfully performing size dependent blood cell insulator based DC dielectrophoresis in PDMS microdevices. In subsequent studies, the optimized microdevice geometry was then used for continuous separation of erythrocytes. The class=SpellE\u3emicrodevice design enabled erythrocyte collection into specific channels based on the cell’s deflection from the high field density region of the obstacle. The channel with the highest concentration of cells is indicative of the ABO blood type of the sample. DC resistance measurement system for quantification of erythrocytes was developed with single PDMS class=SpellE\u3emicrochannel system to be integrated with the DC- class=SpellE\u3eiDEP device developed in this research. This lab-on-a-chip technology application could be applied to emergency situations and naturalcalamities for accurate, fast, and portable blood typing with minimal error

Dielectrophoretic characterization of particles and erythrocytes

Medical lab work, such as blood testing, will one day be near instantaneous and inexpensive via capabilities enabled by the fast growing world of microtechnology. In this research study, sorting and separation of different ABO blood types have been investigated by applying alternating and direct electric fields using class=SpellE\u3edielectrophoresis in microdevices. Poly(dimethylsiloxane) (PDMS) microdevices, fabricated by standard photolithography techniques have been used. Embedded perpendicular platinum (Pt) electrodes to generate forces in AC dielectrophoresis were used to successfully distinguish positive ABO blood types, with O+ distinguishable from other blood types at \u3e95% confidence. This is an important foundation for exploring DC dielectrophoretic sorting of blood types. The expansion of red blood cell sorting employing direct current insulative class=SpellE\u3edielectrophoresis (DC-iDEP) is novel. Here Pt electrodes were remotely situated in the inlet and outlet ports of the microdevice and an insulating obstacle generates the required dielectrophoretic force. The presence of ABO antigens on the red blood cell were found to affect the class=SpellE\u3edielectrophoretic deflection around the insulating obstacle thus sorting cells by type. To optimize the placement of insulating obstacle in the microchannel, COMSOL Multiphysics® simulations were performed. Microdevice dimensions were optimized by evaluating the behaviors of fluorescent polystyrene particles of three different sizes roughly corresponding to the three main components of blood: platelets (2-4 µm), erythrocytes (6-8 µm) and leukocytes (10-15 µm). This work provided the operating conditions for successfully performing size dependent blood cell insulator based DC dielectrophoresis in PDMS microdevices. In subsequent studies, the optimized microdevice geometry was then used for continuous separation of erythrocytes. The class=SpellE\u3emicrodevice design enabled erythrocyte collection into specific channels based on the cell’s deflection from the high field density region of the obstacle. The channel with the highest concentration of cells is indicative of the ABO blood type of the sample. DC resistance measurement system for quantification of erythrocytes was developed with single PDMS class=SpellE\u3emicrochannel system to be integrated with the DC- class=SpellE\u3eiDEP device developed in this research. This lab-on-a-chip technology application could be applied to emergency situations and naturalcalamities for accurate, fast, and portable blood typing with minimal error

Deposition Mechanism and Properties of Thin Polydopamine Films for High Added Value Applications in Surface Science at the Nanoscale

Polydopamine films have been introduced by Messersmith et al. as a possible “versatile” surface functionalization method allowing to coat the surface of almost all known materials even superhydrophobic surfaces. These new kinds of coatings also confer a plethora of functionalities to the coated materials owing to the complex chemistry of the catechol quinone moieties present on the surface of polydopamine. These coatings may hence become an interesting alternative to established surface coatings like self-assembled monolayers and polyelectrolyte multilayered films. In this review, we describe the knowledge acquired in the last 3 years about the deposition mechanisms of polydopamine films, their properties, and various applications in surface science at the nanoscale.Fonds Europeen de Developpement Economique et Regional (Chaptochem Project 2009-02-039-35

Definition of a near real time microbiological monitor for space vehicles

Efforts to identify the ideal candidate to serve as the biological monitor on the space station Freedom are discussed. The literature review, the evaluation scheme, descriptions of candidate monitors, experimental studies, test beds, and culture techniques are discussed. Particular attention is given to descriptions of five candidate monitors or monitoring techniques: laser light scattering, primary fluorescence, secondary fluorescence, the volatile product detector, and the surface acoustic wave detector