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APPLICATION OF POLYELECTROLYTE MULTILAYERS FOR PHOTOLITHOGRAPHIC PATTERNING OF DIVERSE MAMMALIAN CELL TYPES IN SERUM FREE MEDIUM
Title
APPLICATION OF POLYELECTROLYTE MULTILAYERS FOR PHOTOLITHOGRAPHIC PATTERNING OF DIVERSE MAMMALIAN CELL TYPES IN SERUM FREE MEDIUM.
Creator
Dhir, Vipra, Cho, Hyoung Jin, University of Central Florida
Abstract / Description
Integration of living cells with novel microdevices requires the development of innovative technologies for manipulating cells. Chemical surface patterning has been proven as an effective method to control the attachment and growth of diverse cell populations. Patterning polyelectrolyte multilayers through the combination of layer-by-layer self-assembly technique and photolithography offers a simple, versatile and silicon compatible approach that overcomes chemical surface patterning...
Show moreIntegration of living cells with novel microdevices requires the development of innovative technologies for manipulating cells. Chemical surface patterning has been proven as an effective method to control the attachment and growth of diverse cell populations. Patterning polyelectrolyte multilayers through the combination of layer-by-layer self-assembly technique and photolithography offers a simple, versatile and silicon compatible approach that overcomes chemical surface patterning limitations, such as short-term stability and low protein adsorption resistance. In this study, direct photolithographic patterning of PAA/PAAm and PAA/PAH polyelectrolyte multilayers was developed to pattern mammalian neuronal, skeletal and cardiac muscle cells. For all studied cell types, PAA/PAAm multilayers behaved as a negative surface, completely preventing cell attachment. In contrast, PAA/PAH multilayers have shown a cell-selective behavior, promoting the attachment and growth of neuronal cells (embryonic rat hippocampal and NG108-15 cells) to a greater extent, while providing a little attachment for neonatal rat cardiac and skeletal muscle cells (C2C12 cell line). PAA/PAAm multilayer cellular patterns have also shown a remarkable protein adsorption resistance. Protein adsorption protocols commonly used for surface treatment in cell culture did not compromise the cell attachment inhibiting feature of the PAA/PAAm multilayer patterns. The combination of polyelectrolyte multilayer patterns with different adsorbed proteins could expand the applicability of this technology to cell types that require specific proteins either on the surface or in the medium for attachment or differentiation, and could not be patterned using the traditional methods.
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Date Issued
2008
Identifier
CFE0002357, ucf:47783
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0002357
PATTERNED CELL CULTURES FOR HIGH THROUGHPUT STUDIES OF CELL ELECTROPHYSIOLOGY AND DRUG SCREENING APPLICATIONS
Title
PATTERNED CELL CULTURES FOR HIGH THROUGHPUT STUDIES OF CELL ELECTROPHYSIOLOGY AND DRUG SCREENING APPLICATIONS.
Creator
Natarajan, Anupama, Hickman, James, University of Central Florida
Abstract / Description
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...
Show moreOver 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 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 uni-directional 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 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 high-throughput 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.
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Date Issued
2010
Identifier
CFE0003384, ucf:48437
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0003384
Development of human and rodent based in vitro systems toward better translation of bench to bedside in vivo results
Title
Development of human and rodent based in vitro systems toward better translation of bench to bedside in vivo results.
Creator
Berry, Bonnie, Hickman, James, Khaled, Annette, Lambert, Stephen, Sugaya, Kiminobu, University of Central Florida
Abstract / Description
Prospective medicinal compounds progress through multiple testing phases before becoming licensed drugs. Testing of novel compounds includes a preclinical phase where the potential therapeutic is tested in vitro and/or in animal models in vivo to predict its potential efficacy and/or toxicity in humans. The failure of preclinical models to accurately predict human drug responses can lead to potentially dangerous compounds being administered to humans, or potentially beneficial compounds being...
Show moreProspective medicinal compounds progress through multiple testing phases before becoming licensed drugs. Testing of novel compounds includes a preclinical phase where the potential therapeutic is tested in vitro and/or in animal models in vivo to predict its potential efficacy and/or toxicity in humans. The failure of preclinical models to accurately predict human drug responses can lead to potentially dangerous compounds being administered to humans, or potentially beneficial compounds being kept in development abeyance. Moreover, inappropriate choice in model organism for studying disease states may result in pushing forward inappropriate drug targets and/or compounds and wasting valuable time and resources in producing much-needed medications. In this dissertation, models for basic science research and drug testing are investigated with the intention of improving current preclinical models in order to drive drugs to market faster and more efficiently. We found that embryonic rat hippocampal neurons, commonly used to study neurodegenerative disease mechanisms in vitro, take 3-4 weeks to achieve similar, critical ion-channel expression profiles as seen in adult rat hippocampal cultures. We also characterized a newly-available commercial cell line of human induced pluripotent stem cell-derived neurons for their applicability in long-term studies, and used them to develop a more pathologically relevant model of early Alzheimer's Disease in vitro. Finally, we attempted to create an engineered, layered neural network of human neurons to study drug responses and synaptic mechanisms. Utilization of the results and methods described herein will help push forward the development of better model systems for translation of laboratory research to successful clinical human drug trials.
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Date Issued
2015
Identifier
CFE0006261, ucf:51031
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006261