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postgraduate thesis: Epithelial morphogenesis in three-dimensional cell culture system

TitleEpithelial morphogenesis in three-dimensional cell culture system
Authors
Issue Date2014
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Liu, M. [刘梦菲]. (2014). Epithelial morphogenesis in three-dimensional cell culture system. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5387985
AbstractIn 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.
DegreeDoctor of Philosophy
SubjectEpithelial cells
Human cell culture
Dept/ProgramBiochemistry
Persistent Identifierhttp://hdl.handle.net/10722/208611

 

DC FieldValueLanguage
dc.contributor.authorLiu, Mengfei-
dc.contributor.author刘梦菲-
dc.date.accessioned2015-03-13T01:44:08Z-
dc.date.available2015-03-13T01:44:08Z-
dc.date.issued2014-
dc.identifier.citationLiu, M. [刘梦菲]. (2014). Epithelial morphogenesis in three-dimensional cell culture system. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5387985-
dc.identifier.urihttp://hdl.handle.net/10722/208611-
dc.description.abstractIn 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.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.subject.lcshEpithelial cells-
dc.subject.lcshHuman cell culture-
dc.titleEpithelial morphogenesis in three-dimensional cell culture system-
dc.typePG_Thesis-
dc.identifier.hkulb5387985-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineBiochemistry-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b5387985-

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