Patterned Cell Cultures For High Throughput Studies Of Cell Electrophysiology And Drug Screening Applications

Over the last decade, the field of tissue and bio-engineering has seen an increase in the development of in vitro high-throughput hybrid systems that can be used to understand cell function and behavior at the cellular and tissue levels. These tools would have a wide array of applications including for implants, drug discovery, and toxicology, as well as for studying cell developmental behavior and as disease models. Currently, there are a limited number of efficient, functional drug screening assays in the pharmacology industry and studies of cell-surface interactions are complicated and invasive. Most cell physiology studies are performed using conventional patch-clamp techniques or random networks cultured on silicon devices such as Microelectrode Arrays (MEAs) and Field Effect transistors (FETs). The objective of this study was to develop high-throughput in vitro platforms that could be used to analyze cell function and their response to various stimuli. Our hypothesis was that by utilizing surface modification to provide external guidance cues for various cell types and by controlling the cell environment in terms of culture conditions, we could develop an in vitro hybrid platform for sensing and testing applications. Such a system would not only give information regarding the surface effects on the growth and behavior of cells for implant development applications, but also allow for the study of vital cell physiology parameters like conduction velocity in cardiomyocytes and synaptic plasticity in neuronal networks. This study outlines the development of these in vitro high throughput systems that have varied applications ranging from tissue engineering to drug development. We have developed a simple and relatively high-throughput method in order to test the physiological effects of varying iii chemical environments on rat embryonic cardiac myocytes in order to model the degradation effects of polymer scaffolds. Our results, using our simple test system, are in agreement with earlier observations that utilized a complex 3D biodegradable scaffold. Thus, surface functionalization with self-assembled monolayers combined with histological/physiological testing could be a relatively high throughput method for biocompatibility studies and for the optimization of the material/tissue interface in tissue engineering. Traditional multielectrode extracellular recording methods were combined with surface patterning of cardiac myocyte monolayers to enhance the information content of the method; for example, to enable the measurement of conduction velocity, refractory period after action potentials or to create a functional reentry model. Two drugs, 1-Heptanol, a gap junction blocker, and Sparfloxacin, a fluoroquinone antibiotic, were tested in this system. 1-Heptanol administration resulted in a marked reduction in conduction velocity, whereas Sparfloxacin caused rapid, irregular and unsynchronized activity, indicating fibrillation. As shown in these experiments, the patterning of cardiac myocyte monolayers increased the information content of traditional multielectrode measurements. Patterning techniques with self-assembled monolayers on microelectrode arrays were also used to study the physiological properties of hippocampal networks with functional unidirectional connectivity, developed to study the mono-synaptic connections found in the dentate gyrus. Results indicate that changes in synaptic connectivity and strength were chemically induced in these patterned hippocampal networks. This method is currently being used for studying long term potentiation at the cellular level. For this purpose, two cell patterns were optimized for cell migration onto the pattern as demonstrated by time lapse studies, and for iv supporting the best pattern formation and cell survival on these networks. The networks formed mature interconnected spiking neurons. In conclusion, this study demonstrates the development and testing of in vitro highthroughput systems that have applications in drug development, understanding disease models and tissue engineering. It can be further developed for use with human cells to have a more predictive value than existing complex, expensive and time consuming methods

Towards the electrical stimulation of PC12 cells

The electrical stimulation of biological cells has been studied intensively since Galvani’s experiment with a dead frog’s leg muscle. Medical advances such as the pacemaker and the cochlear implants utilize some of the fundamentals of electrical stimulation as a result of these intensive studies. It is also a technique which has been explored in many research laboratories and has been shown to be able to play an important role in neurite outgrowth and the regeneration of transected nerves. Electric stimulation has also been shown to induce the differentiation of PC12 cells, faster than normal chemical means. The pheochromocytoma (PC12) cell line is derived from the pheochromocytoma of the rat adrenal medulla which can be differentiated to display characteristics similar to sympathetic neurons. By being able to induce differentiation faster, this opens the door to using complementary analytical techniques, such as a synchrotron-based Fourier Transform infrared spectromicroscopy to understand some of these molecular processes occurring during the differentiation process. The long term objective of this project is to couple electrical stimulation and Fourier Transform infrared spectromicroscopy to study the electrically induced differentiation process. The primary focus of this work is to develop the methodology and background required to obtain this objective. This thesis focuses primarily on the PC12 cell line which has been reported in literature to differentiate both through chemical means (nerve growth factor, NGF) and electrical stimulation means. Using NGF, PC12 cells were able to show, within 24 hours, signs of initiating the differentiation process. Neurite outgrowths with a mean ± standard deviations of 10.0 ± 9.9, 14.0 ± 12.5, 21.4 ± 26.5, 21.6 ± 38.9 and 40.7 ± 49.1 μm corresponding to 24, 48, 72, 96 and 120 hours of NGF exposure was observed. PC12 cells grown on FTO conductive glass were electrically stimulated with a pulsing sequence of ±50 mV from the resting potential for 1 hour followed by 24 hours of incubation. These cells displayed a mean ± standard deviation neurite length of 9.35 ± 9.19 μm which is similar to PC12 cells exposed to 24 hours of NGF. The results of the electrical stimulation experiments are promising; however more experiments need to be conducted to determine the ideal electrical stimulation parameters to induce differentiation. The promising results also bring us one step closer to coupling FTIR to better understand the differentiation process from a molecular viewpoint

Development of Patterned Self-Assembled Monolayers Toward the Study of Axonal Differentiation

The following work discusses the development of several techniques and new methods for the production of patterned surfaces for protein and cell confinement. These well-defined substrates allow us to study the mechanism of axonal differentiation in neurons confined to a two-dimensional starburst pattern. We utilize self-assembled monolayer: SAM) chemistry in conjunction with microcontact printing to create stable patterned substrates for cell culture. Photolithography is employed in the fabrication of patterned masters, which are used to create elastomeric stamps for microcontact printing. Initially, trichlorosilanes were employed in our patterned SAMs because they react rapidly with glass. These patterned surfaces confined protein and cells to a defined pattern; however, trichlorosilane monomers were difficult to work with because of their extreme reactivity with moisture in the air. An alternative to this highly sensitive system was required to develop stable SAMs. Alkanethiols on gold have traditionally been stable for just 5−7 days in cell culture, but modifications to the linkage between the alkane chain and glycol termination led to the formation of a stable self-assembled monolayer for over five weeks. This is a tremendous advance in the field of SAM chemistry and allows for the study of cellular processes that occur over the course of several weeks. While long-term stability is necessary for the study of developmental events, there are many researchers who do not have the resources to fabricate their own patterned substrates. This led to the development of recyclable, reusable patterned SAMs for cell culture. By utilizing two different methods, either a trypsin analog or detergent, these substrates can be reused up to 11 times over the course of two weeks. This allows investigators to perform several studies on the same patterned substrate, which leads to rapid, reproducible results. The interesting biological question we set out to answer was whether axonal differentiation was an innate process or one that was environmentally determined. We cultured E18 mouse hippocampal neurons on starburst patterned substrates. The starburst consisted of twelve paths of equal width; eleven were short, 20 μm paths and one was longer, ranging from 40 μm to 160 μm. We observed which path the axon grew along by immunostaining for the microtubule-associated tau protein, bound to microtubules in the axon. Our data showed that the axon grows along the long path ~58% of the time for the smallest starburst pattern and the distance a neurite is allowed to extend down a path is linearly correlated to the likelihood of finding the axon on the long path. This points toward axonal differentiation being an environmentally determined process. This combination of photolithography, microcontact printing, and self-assembled monolayer chemistry has led to important advances in the production of stable, patterned substrates for cell culture. We have successfully used this technology to study axonal differentiation and have found that this process is environmentally determined

Design And Fabrication Of Nanodevice For Cell Interfacing

The goal of this thesis is to (a) design and fabricate a nanodevice that interface with cells and (b) optimize neuronal cell culturing protocol. The long term objective of this thesis is to perform intracellular electrical signal recording and stimulation of neuronal cells. To achieve this objective, a nanodevice with “Fin†shaped electrodes was designed that increases the electrode area and conductance so that it reduces the signal loss as shown in the case of traditional circular Nanopillar design. The overarching goal of neuroscience is to target and discover the relationships between the functional connectivity-map of neuronal circuits and their physiological or pathological functions

Development of self-assembled monolayer-based cell culture platform towards fabrication of a three-dimensional bioreactor

The extracellular matrix (ECM) plays an important role in regulating a number of cellular properties and functions like cell differentiation, cell synthesis and degradation, cell viability and proliferation, cell function, and cell aging. Surface modification of planar substrates with self-assembled monolayers (SAMs) is a promising technique to achieve stable ECMs. In this work, substrates such as silicon (Si), gallium arsenide (GaAs) and indium tin oxide (ITO) substrates were modified with SAMS containing amino (-NH2), methyl (-CH3), thiol (-SH) and carboxylic (-COOH) end groups and characterized using contact angle measurements, surface infrared (IR) spectroscopy and atomic force microscopy (AFM). Different cell types such as human dermal fibroblasts (HDFs), mouse stromal mesenchymal stem cells (MSCs), rat brain neurons (RBN), and rat hepatocytes were cultured on these surfaces to develop stable and standard cell culture platforms (CCPs). Contact angle measurements showed that surfaces modified with SAMs containing amino and carboxylic end groups are hydrophilic, methyl terminal group is hydrophobic, and SAM containing thiol end group has an intermediate property. Reflection absorption infrared spectroscopy (RAIRS) and attenuated total reflectance IR (ATR/IR) confirmed the presence of respective SAMs on surfaces. AFM data show that SAMs with methyl and carboxylic group modified surfaces present an average roughness of 1.51 and 2.67 nm, which are higher than 1.01 and 1.1 nm obtained for SAMs containing amino and thiol end groups. For cell culture studies on SAM-modified surfaces, viability was assessed using the LIVE/DEAD® assay, proliferation by the MTT assay, while phenotypic maintenance was monitored by immuohistochemical detection of Type I collagen. Morphological responses of the cells were studied using phase contrast and fluorescence imaging to document changes in cell shape and properties. Based on their viability, proliferation and phenotype, HDF cells preferred the substrates in the following order: ITO-ODT \u3e Si-APTES \u3e ITO \u3e Si \u3e GaAs-ODT \u3e GaAs. MSCs grew well on all SAM-modified surfaces with highest proliferation observed on thiol (-SH) terminated ITO substrates. For neuronal cells, addition of 1% serum initially to the cell suspension maintained their viability on methyl and amino modified ITO substrates by neutralizing the effects of dimethyl sulfoxide (DMSO). Neurons preferred amino over methyl terminated SAMs on ITO. (Abstract shortened by UMI.

Design And Fabrication Of Nanodevice For Cell Interfacing

The goal of this thesis is to (a) design and fabricate a nanodevice that interface with cells and (b) optimize neuronal cell culturing protocol. The long term objective of this thesis is to perform intracellular electrical signal recording and stimulation of neuronal cells. To achieve this objective, a nanodevice with “Fin†shaped electrodes was designed that increases the electrode area and conductance so that it reduces the signal loss as shown in the case of traditional circular Nanopillar design. The overarching goal of neuroscience is to target and discover the relationships between the functional connectivity-map of neuronal circuits and their physiological or pathological functions

Surface chemistry modification of glass and gold for low density neural cell culture

Surface chemical modifications are presented for supporting primary neurons in culture. The initial substrates for culture were glass and gold. The surface modifications were based on self assembled monolayer (SAM) approaches. Glass surfaces were initially modified by silanisation with either 3-aminopropyltrimethoxysilane (APTMS) or 3-aminopropyldimethylethoxysilane (APDES), to present amino-terminated surfaces. Gold surfaces were initially modified by thiol SAMs of either 11-amino-1-undecanethiol (AUT) or a peptide fragment of laminin (PA22-2), to present an amino- or peptide-terminated surface respectively. The amine-terminated surfaces of both glass and gold were subject to further modification. A heterobifunctional linker, containing a polyethylene glycol (PEG) spacer, was used to couple the peptide PA22-2 to the amino-terminated surfaces. Surface modifications were characterised using WCA, XPS and ToF-SIMS. The heterobifunctional linker bound homogeneously across the AUT SAM surface, however the linker was not distributed evenly on either of the amino silanisations of glass. Primary neurons from dissociated embryonic rat hippocampi were cultured on the modified glass and gold surfaces. The cell viability was measured during a 3 week long culture using calcein and ethidium homodimer fluorescence. Neuronal cell cultures were viable on all the gold surface modifications. The only viable glass surface was a control surface of adsorbed poly-l-lysine (PLL) on glass. Cell viability on the AUT and the Peptide-PEG-AUT modified gold surfaces was equivalent to the PLL coated glass. Inclusion of the PEG linker reduced protein adsorption from the media to the peptide modified gold surface, allowing cells to recognise the peptide rather than an adsorbed protein layer and improving their viability. The presented gold surface modifications provide suitable substrates for neural cultures which can be used in existing applications for investigating neural activity, such as; multi-electrode arrays, micro-fluidics devices, and surface plasmon resonance

Surface chemistry modification of glass and gold for low density neural cell culture

Surface chemical modifications are presented for supporting primary neurons in culture. The initial substrates for culture were glass and gold. The surface modifications were based on self assembled monolayer (SAM) approaches. Glass surfaces were initially modified by silanisation with either 3-aminopropyltrimethoxysilane (APTMS) or 3-aminopropyldimethylethoxysilane (APDES), to present amino-terminated surfaces. Gold surfaces were initially modified by thiol SAMs of either 11-amino-1-undecanethiol (AUT) or a peptide fragment of laminin (PA22-2), to present an amino- or peptide-terminated surface respectively. The amine-terminated surfaces of both glass and gold were subject to further modification. A heterobifunctional linker, containing a polyethylene glycol (PEG) spacer, was used to couple the peptide PA22-2 to the amino-terminated surfaces. Surface modifications were characterised using WCA, XPS and ToF-SIMS. The heterobifunctional linker bound homogeneously across the AUT SAM surface, however the linker was not distributed evenly on either of the amino silanisations of glass. Primary neurons from dissociated embryonic rat hippocampi were cultured on the modified glass and gold surfaces. The cell viability was measured during a 3 week long culture using calcein and ethidium homodimer fluorescence. Neuronal cell cultures were viable on all the gold surface modifications. The only viable glass surface was a control surface of adsorbed poly-l-lysine (PLL) on glass. Cell viability on the AUT and the Peptide-PEG-AUT modified gold surfaces was equivalent to the PLL coated glass. Inclusion of the PEG linker reduced protein adsorption from the media to the peptide modified gold surface, allowing cells to recognise the peptide rather than an adsorbed protein layer and improving their viability. The presented gold surface modifications provide suitable substrates for neural cultures which can be used in existing applications for investigating neural activity, such as; multi-electrode arrays, micro-fluidics devices, and surface plasmon resonance

Moving live dissociated neurons with an optical tweezer

The use of an optical tweezer for moving dissociated neurons was studied. The main features of the tweezers are outlined as well as the general principles of its operation. Infrared beams at 980 and 1064 nm were used, focused so as to make a trap for holding neurons and moving them. Absorption by cells at those wavelengths is very small. Experiments were done to evaluate nonsticky substrate coatings, from which neurons could be easily lifted with the tweezers. The maximum speed of cell movement as a function of laser power was determined. Detailed studies of the damage to cells as a function of beam intensity and time of exposure were made. The 980 nm beam was much less destructive, for reasons that are not understood, and could be used to safely move cells through distances of millimeters in times of seconds. An illustrative application of the use of the tweezers to load neurons without damage into plastic cages on a glass substrate was presented. The conclusion is that optical tweezers are an accessible and practical tool for helping to establish neuron cultures of cells placed in specific locations