In human body, the most common structures formed by epithelial cells are hollow cysts or tubules. The key feature of the cysts and tubules is the central lumen, which is lined by epithelial cell sheets. The central lumen allows material exchange, thus it is indispensable for the proper function of the epithelial tissue. In order to understand the way that the epithelial cells form highly specialized structure, an in vitro three-dimensional (3D) culture system was established. The Caco-2 cells were embedded in reconstituted basement membrane termed matrigel, whose biochemical constitution and physical properties were similar with the in vivo environment. The Caco-2 cells in matrigel spontaneously formed spherical multi-cell cysts, which could continuously expand. The confocal imaging and reconstruction technique helped understand the cyst structure and its formation process. The cysts developed central lumen surrounded by a layer of polarized cells. The apical domain of the cells faced the lumen, while the basal domain attached to the extracellular matrix. In the mature cysts, fluid was secreted by the cells around the lumen at the apical domain, and accumulated in the central lumen. The laser burning experiment showed that the intraluminal pressure was higher than the outer environment. The intact cell sheet was required to keep the engorged morphology of the cysts. The tension of the cell layer balanced with the intraluminal pressure. To investigate the effect of pressure on cyst development, the cysts were treated with cholera toxin, which could increase intraluminal pressure through promoting apical secretion. The time-lapse images showed that under cholera toxin treatment, the expansion of the cysts was accelerated. The high intraluminal pressure led to shape change of thecells, followed by increase in cell proliferation rate. Cholera toxin itself could not promote cell growth. In the3D cultured cysts, it was the increased intraluminal pressure that directly induced the acceleration of cell proliferation. It indicated that not only biochemical signals, but also mechanical force, contributed to epithelial morphogenesis. The mechanical stimulation could be converted into biochemical signals, further affect cell behavior. In response to mechanical stimulation, the focal adhesion kinase was activated in the cells around the cyst lumen. Furthermore, the microarray analysis suggested that multiple signaling pathways were altered under intraluminal pressure stimulation, including the pathways related to cytoskeleton organization, cell cycle and cell adhesion. Taken together, comparing with the conventional two-dimensional cell culture on rigid surface, the three-dimensional culture system provided the cells a more physiological environment. The 3D culture system allows the epithelial cells to form well-organized hollow structure. It is a convenient model for investigating the process and mechanism of epithelial morphogenesis. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Thesis supervisor: Maureen D. Donovan. Includes bibliographic references (p. 239-247).

Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliographical references.

published_or_final_version / Anatomy / Master / Master of Philosophy

This project developed methodology for in vitro toxicity assessment of contaminated sites using the Promega?? MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay performed on human cells (HepG2 and Skin fibroblasts). The project included the development of a method for extracting contaminants from soil based on leaching and centrifugation. A number of solvents and surfactants were assessed for their suitability as extracting agents. The Zwitterionic surfactant CHAPS ({3[(3-Cholamidopropyl) dimethylammonio] propanesulphonic acid}), which is an irritant in vivo, was found suitable for in vitro toxicity assessment applications. CHAPS was found to be the least toxic surfactant in vitro when tested on skin fibroblasts (NOEC: 1800??577 ppm, IC50: 4000??577 ppm) and HepG2 cells (NOEC: 833??289 ppm, IC50: 5300??287 ppm). The chosen surfactant was used in three different methods for extraction of Toluene and Xylene spiked in 2 g and 10g soil. The combination comprising of 0.1% (s/w) CHAPS and cosolvent 1% (w/w) Isopropanol, at their respective NOEC (No Observed Effective Concentration) toxicity values, showed good recovery of the nonpolar organic compounds in comparison to the recovery by 0.1% CHAPS and 0.5% CHAPS. The study found additive interactions to be the most common form of toxicity for 16 concentration combinations of Formaldehyde (polar), Toluene and Xylene (nonpolar) when compared to predicted toxicity (R2=0.943, P&lt0.0001). When assessing the in vitro toxicity of unknown (blind) contaminated soil samples, the Hazard Index (HI) predicted from the chemical analyses results showed a relatively good correlation (R2&gt0.7062, n=26) when compared to the experimental toxicity results on HepG2 cells. Furthermore, the comparison of Australian Health Investigation Levels (HIL) with in vitro toxicity testing gave similar correlation (R2&gt0.6882, n=26) on HepG2 cells. The overall project suggests the potential application of the zwitterionic surfactant (CHAPS) in sampling contaminants from soils in an in vitro toxicity assessment. This study demonstrates the application of in vitro toxicity assessment using human cells for the prediction of toxic risk as a sentinel to human toxicity from a contaminated site.

Univ., Diss.--Göteborg, 2005. / Literaturverz. S. [289] - 313.

No description available.

This project developed methodology for in vitro toxicity assessment of contaminated sites using the Promega?? MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay performed on human cells (HepG2 and Skin fibroblasts). The project included the development of a method for extracting contaminants from soil based on leaching and centrifugation. A number of solvents and surfactants were assessed for their suitability as extracting agents. The Zwitterionic surfactant CHAPS ({3[(3-Cholamidopropyl) dimethylammonio] propanesulphonic acid}), which is an irritant in vivo, was found suitable for in vitro toxicity assessment applications. CHAPS was found to be the least toxic surfactant in vitro when tested on skin fibroblasts (NOEC: 1800??577 ppm, IC50: 4000??577 ppm) and HepG2 cells (NOEC: 833??289 ppm, IC50: 5300??287 ppm). The chosen surfactant was used in three different methods for extraction of Toluene and Xylene spiked in 2 g and 10g soil. The combination comprising of 0.1% (s/w) CHAPS and cosolvent 1% (w/w) Isopropanol, at their respective NOEC (No Observed Effective Concentration) toxicity values, showed good recovery of the nonpolar organic compounds in comparison to the recovery by 0.1% CHAPS and 0.5% CHAPS. The study found additive interactions to be the most common form of toxicity for 16 concentration combinations of Formaldehyde (polar), Toluene and Xylene (nonpolar) when compared to predicted toxicity (R2=0.943, P&lt0.0001). When assessing the in vitro toxicity of unknown (blind) contaminated soil samples, the Hazard Index (HI) predicted from the chemical analyses results showed a relatively good correlation (R2&gt0.7062, n=26) when compared to the experimental toxicity results on HepG2 cells. Furthermore, the comparison of Australian Health Investigation Levels (HIL) with in vitro toxicity testing gave similar correlation (R2&gt0.6882, n=26) on HepG2 cells. The overall project suggests the potential application of the zwitterionic surfactant (CHAPS) in sampling contaminants from soils in an in vitro toxicity assessment. This study demonstrates the application of in vitro toxicity assessment using human cells for the prediction of toxic risk as a sentinel to human toxicity from a contaminated site.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 97-112) Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 97-112) Also available online.