The environment encountered by a single cell in vivo is a complex and dynamic system that is often simplified experimentally via ex vivo and in vitro methods. As our understanding of cell response in these basic environments grows, there is a corresponding need for techniques that modify traditional cell culture in ways that better mimic the complexities of in vivo systems. This dissertation examines how the three dimensional (3D) properties of a focused pulsed laser can be incorporated within existing techniques to dynamically manipulate these microenvironments in the presence of single cells. As a modification on existing microfluidic technology for chemically dosing cells, it is shown how a cost-effective microchip laser can be used to ablate microscopic pores in a thin, biocompatible polymer membrane. These pores serve as conduits for introducing dosing reagents in close proximity to cultured cells combining subcellular resolution with spatial and temporal control. Because reagent flow is physically separated from the cell-culture flow chamber by this polymer membrane, the geometry of the reagent flow cell can be altered to accommodate multiple reagents flowing in parallel with minimal mixing due to the laminar flow characteristics of microfluidic devices. By manipulating reagent flow, a single cell can be dosed at opposing ends by distinct reagents or by defined, stable gradients of a single reagent. Additionally, these dosing streams can be switched with subsecond temporal resolution or dynamically mixed to study potential synergistic or antagonistic effects. To define the physical environment surrounding small populations of cells, an existing platform for mask-directed multiphoton lithography is used to create biocompatible protein-based microstructures for studying cancer-cell migration and invasion in physically confined regions. In these studies, a variety of 3D shapes incorporating spatial gradients are examined with invasive cell types. Additionally, these methods have been modified to allow for in situ fabrication of gelatin microstructures with 3D resolution around suspended somatic cells by covalently binding a photosensitizing molecule to the protein prior to fabrication. The architecture of these microstructures is designed to provide a variety of 3D confinement scenarios with biological relevance. / text

No description available.

University of Macau / Institute of Chinese Medical Sciences

The fabrication, motion and behavior of small droplets are subjects under considerable current study. The possible applications include using droplets as actuators to enhance mixing, as chemical reactors and the formation of emulsions. Microfluidics provides a convenient means of producing droplets at the micro scale. The study is currently dominated by spherical systems where droplets are consistently spherical in nature. Various methods and geometries have been tested for fabricating these droplets but little research has been conducted towards producing non-circular droplets. While the fabrication of non-spherical droplets has been reported before control over their shape remains difficult to achieve. In this thesis, we present a method to fabricate droplets using shear focusing in an oil medium alternatively from two channels facing each other. The droplets produced are non-circular in shape, and their shape dynamically alters as they travel in the microfluidic channel. The size of the droplets can be controlled by the ratio of oil and water flow rates. Microscopic images have been presented that show the non-spherical shape of the droplets at the point of fabrication. Images taken at two points further along the microfluidic channel show how the shapes of these droplets change as they travel in the channel. There were three regimes of droplet shapes, circular, triangular and rectangular shapes that were determined by the packing ratio of water droplet in oil phase in microfluidic channels. All droplets formed in this experiment were monodispersed.

Mixing of fluids at the microscale poses a variety of challenges, many of which arise from the fact that molecular diffusion is the dominant transport mechanism in the laminar flow regime. The unfavorable combination of low Reynolds numbers and high Péclet numbers implies that cumbersomely long microchannels are required to achieve efficient levels of micromixing. Although considerable progress has been made toward overcoming these limitations (e.g., exploiting chaotic effects), many techniques employ intricate 3-D flow networks whose complexity can make them difficult to build and operate. In this research, we show that enhanced micromixing can be achieved using topologically simple and easily fabricated planar 2-D microchannels by simply introducing curvature and changes in width in a prescribed manner. This is accomplished by harnessing a synergistic combination of (i) Dean vortices that arise in the vertical plane of curved channels as a consequence of an interplay between inertial, centrifugal, and viscous effects, and (ii) expansion vortices that arise in the horizontal plane due to an abrupt increase in a conduit’s cross-sectional area. We characterize these effects using top-view imaging of aqueous streams labeled with tracer dyes and confocal microscopy of aqueous fluorescent dye streams, and by observing binding interactions between an intercalating dye and double-stranded DNA. These mixing approaches are versatile, scalable, and can be straightforwardly integrated as generic components in a variety of lab-on-a-chip systems.

Thesis (M.A.Sc.) - Simon Fraser University, 2007. / Theses (School of Engineering Science) / Simon Fraser University. Senior supervisor: Dr. Bonnie Gray -- School of Engineering Science. Also issued in digital format and available on the World Wide Web.

published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

No description available.

Exosomes are microvesicles that are released from several different types of cells. Exosomes are thought to play an important role in functions such as immune regulation and coagulation; however their full functionality is not completely understood. Current research has started to explore their potential utilization in gene therapy and drug delivery. Their derivation from different proteins and RNA make them a versatile transport target in microbiology research. Although exosomes are being increasingly used in current research for gene therapy applications, the actual mechanism is unknown once the exosomes are taken into the cells. Using microfluidic channels, the entire process of exosome uptake can be imaged and monitored. The design of the microfluidic device allows for the manipulation of cellular flow and imitates the real flow of cells during exosome uptake and interaction. The microfluidic device is made from a mold using polydimethylsiloxane (PDMS) and the channels are coated with fibronectin for the cells to adhere to. The device is plasma bonded to a thin sheet of PDMS, incubated, and then left to cure. Because of its ability to grow quickly and efficiently in less-than-ideal conditions, hey ovarian cancer cells are used to seed the device. The hey cells are seeded at a density between five million and 10 million cells in the device, and fresh media is pumped through the device. The cells are left to adhere and proliferate for between 24 hours while fresh media is passed through the device in the 37 degrees C incubator. The hey cells are dyed using a DAPI fluorescent stain which causes the hey cells to illuminate blue fluorescence. Exosomes that are stained with PKH to illuminate green fluorescence are then seeded into the channels, and images are taken using confocal microscopy at several time points. The images showing blue fluorescent hey cells and green fluorescent exosomes are overlayed to show the exosomes uptake into the hey cells. Several images are taken across approximately a 20-minute time period to show the interaction between the exosomes and the cells in the channels.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 146-151).