Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

We describe a simple, sensitive, nonradioactive, relatively rapid and relatively inexpensive protocol to measure DNA synthesis in cultured cells by a chemiluminescent bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA). We show that it exhibits similar sensitivity and activity as traditional 3H-thymidine incorporation assays and a commercial chemiluminescent BrdU ELISA kit when tested in commonly used cell lines, such as NIH 3T3 cells, mink lung epithelial cells (Mv1LU), and baby hamster kidney (BHK-21) fibroblasts. This assay also exhibits a wider dynamic range than colorimetric BrdU ELISA methods. Besides being a viable, nonradioactive alternative to 3H-thymidine incorporation assays, our BrdU ELISA is less expensive than a commercial chemiluminescent BrdU ELISA kit.

Precise control over microbial cell growth conditions could enable detection of minute phenotypic changes, which would improve our understanding of how genotypes are shaped by adaptive selection. Although automated cell- culture systems such as bioreactors offer strict control over liquid culture conditions, they often do not scale to high-throughput or require cumbersome redesign to alter growth conditions. I report the design and validation of eVOLVER, a scalable DIY framework that can be configured to carry out high- throughput growth experiments in molecular evolution, systems biology, and microbiology. I perform high-throughput evolution of yeast across systematically varied population density niches to show how eVOLVER can precisely characterize adaptive niches. I describe growth selection using time-varying temperature programs on a genome-wide yeast knockout library to identify strains with altered sensitivity to changes in temperature magnitude or frequency. Inspired by large-scale integration of electronics and microfluidics, I also demonstrate millifluidic multiplexing modules that enable multiplexed media routing, cleaning, vial-to-vial transfers and automated yeast mating.

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The goal of this study is to design a protocol for the adherent cell culture within a novel microfluidic device. Microscale cell culture protocols were developed for loading cells using poly-L-lysine to enhance adherent cell culture of murine derived NIH 3T3 fibroblasts. This work sought to develop a method for adherent microculture by examining various sterilization, surface treatment, and seeding techniques. Using a vacuum suction loading technique, air plasma treatment and a poly-L-lysine surface treatment adherent cell culture was observed within the device. The work presented here is part of a collaborative effort that aims to develop protocols for the electrical and optical characterization of cell culture within a novel microfluidic device.

Since bone tissues grow in intimate contact with implant surfaces in vivo, there is a need to investigate how bone cells respond to mechanical loads adjacent to implants under well characterized loading conditions that stimulate the bone-implant surface. Thus, the objective of this study was to demonstrate an effective means for applying known, uniform, cyclic strain to cells growing on implant materials in vitro. A cell culture strain plate device was developed based on the application of the four-point bending principle. The device uses a small electric motor to drive belts attached to shafts which turn a set of cams. The cams are attached to pins which connect to a titanium plate which rests over arched supports. When deflected and depending on which set of cams are used, strains generated range from around 200 to 1000 ìstrain. UMR-106 osteoblast-like cells were cultured on the titanium plate, and the plate was deflected at three strain magnitudes at 1.5 Hz for durations of 4 and 24 hours. Strain gages recorded average maximum strain levels of 182 ± 3, 366 ± 9, and 984 ± 7µstrain. The strain device, with attached cells, was tested in an amiable bioenvironment. Results from strain gages indicated a uniform strain field existed within the center region of the plate and culture area. Cells in the test plates stained viable, exhibited similar morphology to controls, and were assayed for alkaline phosphatase (ALP) activity, total protein production, and calcium deposition. Results also indicated that stretched cells exhibited increases in proliferation, as well as changes in ALP activity vs. unstrained controls. Thus, the device was successful in distinguishing differences in cell response to mechanical perturbations and may be used to investigate how cells respond to strains at implant-bone interfaces.

Bone and bone cells have been shown to respond to mechanical forces placed upon them. Particularly, strain plays an important role in osteogenic differentiation of marrow cells around artificial implants in bone. These strains, depending on their magnitude, duration, and repetition, can alter the proliferation and matrix synthesis of osteoblasts. To test how strain parameters influence osteoblast behavior, a four-point bending apparatus was used to impose cyclic strain on osteogenic stem cells isolated from rats and seeded on titanium plates. Cells were stimulated at 1 Hz for 15 minutes daily and compared to an unstrained control. Stimulation occurred at two magnitudes: 400 and 1000 micro-strain, and three levels of repetition: one, three, and five consecutive days. DNA, protein, alkaline phosphatase, and calcium levels were measured to determine the proliferation and matrix synthesis activity of the cells. No statistically significant effect was found for the tested parameters under these conditions.

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