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postgraduate thesis: Multiphoton microfabrication-based cell niche screening : phenotype maintenance of nucleus pulposus cells as an example

TitleMultiphoton microfabrication-based cell niche screening : phenotype maintenance of nucleus pulposus cells as an example
Authors
Advisors
Advisor(s):Chan, BP
Issue Date2019
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Yip, C. [葉智鴻]. (2019). Multiphoton microfabrication-based cell niche screening : phenotype maintenance of nucleus pulposus cells as an example. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.
AbstractMany cells lose their phenotype and functions upon monolayer cultures on rigid and flat culture dishes, referring to a process called dedifferentiation. This may lead to physiologically-irrelevant results in cell research and pharmaceutical industry. This is owing to the fact that the in-vitro condition differs significantly from the physiological microenvironment of the cells in native tissue. As a result, cell niche engineering technologies able to screen and reconstitute cell niche factors for enhanced cell phenotype maintenance in vitro is critical to unbiased niche design process. Our lab has developed a multiphoton microfabrication and micropatterning (MMM) technology and microfabricated a wide range of protein microstructures. Here, we employed this technology to develop a multiplex bottom-up cell niche factor screening platform for phenotype maintenance, using bovine nucleus pulposus cell (bNPC), which has a critical role in maintaining the nucleus pulposus (NP) tissue integrity and the intervertebral disc (IVD) functions, as an example. Chapter 1 starts with an introduction to the phenomenon of monolayer-induced cell dedifferentiation. The significance of cell niche and its constituent components, together with the existing micropatterning technologies for cell niche engineering studies, particularly the MMM technology, have been discussed. Meanwhile, the shift from macro-scale to micro-scale screening in recent years has encouraged the development of mature image analysis techniques. The overall design of this study is also discussed. Chapters 2 and 3 presented a preparation phase before screening. Chapter 2 presents technological development in fabricating complex 3D continuous protein microstructures, enabling fabrication of topological features as one independent niche factor to be used in the screening stage. Chapter 3 determines the screening parameters which are closely related to the bNPC dedifferentiation process. The phenotypes of native NP tissues and cells, together with the dedifferentiation process upon monolayer culture, were examined to rationalize screening readouts, cells and time frame for the subsequent screening experiments. The cell niche factor screening was conducted in two stages. Chapter 4 is a first-line screening investigating the effects of individual cell niche factors on bNPC phenotype marker expressions. Among numerous mechanical, topological and extracellular matrix (ECM) cell niche factors, stiffness at low level, topological features including micropillar arrays (MPA), fiber-bead microstructure (FB), and ECM including laminin and vitronectin were found to better maintain the bNPC marker expressions, and thus shortlisted for cell niche design in next chapter. Chapter 5 is the second line of screening where the niche factors shortlisted previously were combined to form complex niche designs. Indeed, these complex niches significantly increased bNPC marker expressions, suggesting that these combinations of niche factors could serve as phenotype-maintaining niches for bNPCs. The last chapter (Ch.6) discusses the prospect of the technology, screening approach and the subsequent works. Taken together, the cell niche screening pipeline using MMM technology was developed and the cell niches supportive to bNPC phenotype maintenance have been identified accordingly. This screening platform can be used to optimize the culture conditions for many cell types other than the current bNPC example and investigate biological problems in addition to phenotype maintenance of somatic cells.
DegreeDoctor of Philosophy
SubjectCell culture
Microfabrication
Dept/ProgramMechanical Engineering
Persistent Identifierhttp://hdl.handle.net/10722/279267

 

DC FieldValueLanguage
dc.contributor.advisorChan, BP-
dc.contributor.authorYip, Chi-hung-
dc.contributor.author葉智鴻-
dc.date.accessioned2019-10-24T08:28:41Z-
dc.date.available2019-10-24T08:28:41Z-
dc.date.issued2019-
dc.identifier.citationYip, C. [葉智鴻]. (2019). Multiphoton microfabrication-based cell niche screening : phenotype maintenance of nucleus pulposus cells as an example. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.-
dc.identifier.urihttp://hdl.handle.net/10722/279267-
dc.description.abstractMany cells lose their phenotype and functions upon monolayer cultures on rigid and flat culture dishes, referring to a process called dedifferentiation. This may lead to physiologically-irrelevant results in cell research and pharmaceutical industry. This is owing to the fact that the in-vitro condition differs significantly from the physiological microenvironment of the cells in native tissue. As a result, cell niche engineering technologies able to screen and reconstitute cell niche factors for enhanced cell phenotype maintenance in vitro is critical to unbiased niche design process. Our lab has developed a multiphoton microfabrication and micropatterning (MMM) technology and microfabricated a wide range of protein microstructures. Here, we employed this technology to develop a multiplex bottom-up cell niche factor screening platform for phenotype maintenance, using bovine nucleus pulposus cell (bNPC), which has a critical role in maintaining the nucleus pulposus (NP) tissue integrity and the intervertebral disc (IVD) functions, as an example. Chapter 1 starts with an introduction to the phenomenon of monolayer-induced cell dedifferentiation. The significance of cell niche and its constituent components, together with the existing micropatterning technologies for cell niche engineering studies, particularly the MMM technology, have been discussed. Meanwhile, the shift from macro-scale to micro-scale screening in recent years has encouraged the development of mature image analysis techniques. The overall design of this study is also discussed. Chapters 2 and 3 presented a preparation phase before screening. Chapter 2 presents technological development in fabricating complex 3D continuous protein microstructures, enabling fabrication of topological features as one independent niche factor to be used in the screening stage. Chapter 3 determines the screening parameters which are closely related to the bNPC dedifferentiation process. The phenotypes of native NP tissues and cells, together with the dedifferentiation process upon monolayer culture, were examined to rationalize screening readouts, cells and time frame for the subsequent screening experiments. The cell niche factor screening was conducted in two stages. Chapter 4 is a first-line screening investigating the effects of individual cell niche factors on bNPC phenotype marker expressions. Among numerous mechanical, topological and extracellular matrix (ECM) cell niche factors, stiffness at low level, topological features including micropillar arrays (MPA), fiber-bead microstructure (FB), and ECM including laminin and vitronectin were found to better maintain the bNPC marker expressions, and thus shortlisted for cell niche design in next chapter. Chapter 5 is the second line of screening where the niche factors shortlisted previously were combined to form complex niche designs. Indeed, these complex niches significantly increased bNPC marker expressions, suggesting that these combinations of niche factors could serve as phenotype-maintaining niches for bNPCs. The last chapter (Ch.6) discusses the prospect of the technology, screening approach and the subsequent works. Taken together, the cell niche screening pipeline using MMM technology was developed and the cell niches supportive to bNPC phenotype maintenance have been identified accordingly. This screening platform can be used to optimize the culture conditions for many cell types other than the current bNPC example and investigate biological problems in addition to phenotype maintenance of somatic cells.-
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.rightsThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.-
dc.subject.lcshCell culture-
dc.subject.lcshMicrofabrication-
dc.titleMultiphoton microfabrication-based cell niche screening : phenotype maintenance of nucleus pulposus cells as an example-
dc.typePG_Thesis-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineMechanical Engineering-
dc.description.naturepublished_or_final_version-
dc.date.hkucongregation2019-
dc.identifier.mmsid991044158739703414-

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