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postgraduate thesis: Multiphoton crosslinking-based protein micropatterning : a platform to study interactions between cells and matrix niche

TitleMultiphoton crosslinking-based protein micropatterning : a platform to study interactions between cells and matrix niche
Authors
Advisors
Advisor(s):Chan, BP
Issue Date2018
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Huang, N. [黃楠]. (2018). Multiphoton crosslinking-based protein micropatterning : a platform to study interactions between cells and matrix niche. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.
AbstractCell niche is the microenvironment providing the cell with both biochemical signals, e.g., the extracellular matrix (ECM) proteins, and mechanical signals, e.g., the elastic modulus, that can support the cell survival, and determine the cell fates. Thus, the ability to reconstitute biomimetic cell niche would potentially impact on tissue engineering and regenerative medicine. Our lab has previously developed a two-photon excitation based crosslinking technology to reconstitute the 3D protein-based biomimetic cell niche. Specifically, bovine serum albumin (BSA) was used as the substrate material and rose Bengal (RB) was used as the photosensitizer of the crosslinking process. However, existing microfabrication technologies such as microcontact printing, soft lithography, and replica-molding have several drawbacks such as time-consuming fabrication process and low fabrication resolution. Our technology showed several advantages including high-resolution 3D reconstitution, excellent biocompatibility, precise and spatial control of the properties of the cell niche and simple “write and seed” step for cell culture, over existing technologies. Here, we aim to firstly study the relationship between the fabrication parameters and the mechanical properties, particularly the elastic modulus of BSA microstructures. In details, different combinations of fabrication parameters were used to define the optimal set of parameters for fabrication of protein-based cell niche. In the meantime, we used transmission electron microscopy (TEM) to define the inverse association between porosity and elastic modulus of BSA microstructures. These results helped us to have a better control of the mechanical properties in the reconstitution of a biomimetic cell niche. Second, we aim to incorporate ECM proteins as the biochemical signals to reconstitute the biomimetic cell niche by utilizing a two-step crosslinking method. Technically, we fabricated BSA microstructures and then crosslinked ECM proteins to these microstructures. Here we used the laser power and scan cycles to precisely control the local density of ECM proteins, while multiple types of ECM proteins were spatially and individually crosslinked in a single cell niche through replacing the solutions of ECM protein for crosslinking. Immunofluorescence staining results demonstrated that ECM proteins were successfully crosslinked to the surface of BSA microstructures. Third, we aim to decouple the biochemical and mechanical properties of the reconstituted biomimetic cell niche, which was further verified by Atomic force microscopy (AFM) testing and immunofluorescence staining. Integrin, pFAK, actin, nucleus of human mesenchymal stem cell (hMSC) were labeled for the cell-matrix interaction study. The frequency map of integrin and pFAK showed the hMSCs’ preference of certain ECM protein local density and higher elastic modulus. It also indicated that different types of ECM protein possessed different cell behaviors, for example, cell attachment, cell survival and cell spreading during cell-matrix interactions. We set up a two-photon microfabrication platform for a precise and spatial control of the biochemical and the mechanical properties independently in a 3D protein-based biomimetic cell niche. We also established a single-cell based cell niche platform by using this technology. Our further studies will focus on incorporating more bioactive signals to better mimic the native 3D cell niche microenvironment.
DegreeDoctor of Philosophy
SubjectExtracellular matrix proteins
Microfabrication
Dept/ProgramMechanical Engineering
Persistent Identifierhttp://hdl.handle.net/10722/255459

 

DC FieldValueLanguage
dc.contributor.advisorChan, BP-
dc.contributor.authorHuang, Nan-
dc.contributor.author黃楠-
dc.date.accessioned2018-07-05T07:43:38Z-
dc.date.available2018-07-05T07:43:38Z-
dc.date.issued2018-
dc.identifier.citationHuang, N. [黃楠]. (2018). Multiphoton crosslinking-based protein micropatterning : a platform to study interactions between cells and matrix niche. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.-
dc.identifier.urihttp://hdl.handle.net/10722/255459-
dc.description.abstractCell niche is the microenvironment providing the cell with both biochemical signals, e.g., the extracellular matrix (ECM) proteins, and mechanical signals, e.g., the elastic modulus, that can support the cell survival, and determine the cell fates. Thus, the ability to reconstitute biomimetic cell niche would potentially impact on tissue engineering and regenerative medicine. Our lab has previously developed a two-photon excitation based crosslinking technology to reconstitute the 3D protein-based biomimetic cell niche. Specifically, bovine serum albumin (BSA) was used as the substrate material and rose Bengal (RB) was used as the photosensitizer of the crosslinking process. However, existing microfabrication technologies such as microcontact printing, soft lithography, and replica-molding have several drawbacks such as time-consuming fabrication process and low fabrication resolution. Our technology showed several advantages including high-resolution 3D reconstitution, excellent biocompatibility, precise and spatial control of the properties of the cell niche and simple “write and seed” step for cell culture, over existing technologies. Here, we aim to firstly study the relationship between the fabrication parameters and the mechanical properties, particularly the elastic modulus of BSA microstructures. In details, different combinations of fabrication parameters were used to define the optimal set of parameters for fabrication of protein-based cell niche. In the meantime, we used transmission electron microscopy (TEM) to define the inverse association between porosity and elastic modulus of BSA microstructures. These results helped us to have a better control of the mechanical properties in the reconstitution of a biomimetic cell niche. Second, we aim to incorporate ECM proteins as the biochemical signals to reconstitute the biomimetic cell niche by utilizing a two-step crosslinking method. Technically, we fabricated BSA microstructures and then crosslinked ECM proteins to these microstructures. Here we used the laser power and scan cycles to precisely control the local density of ECM proteins, while multiple types of ECM proteins were spatially and individually crosslinked in a single cell niche through replacing the solutions of ECM protein for crosslinking. Immunofluorescence staining results demonstrated that ECM proteins were successfully crosslinked to the surface of BSA microstructures. Third, we aim to decouple the biochemical and mechanical properties of the reconstituted biomimetic cell niche, which was further verified by Atomic force microscopy (AFM) testing and immunofluorescence staining. Integrin, pFAK, actin, nucleus of human mesenchymal stem cell (hMSC) were labeled for the cell-matrix interaction study. The frequency map of integrin and pFAK showed the hMSCs’ preference of certain ECM protein local density and higher elastic modulus. It also indicated that different types of ECM protein possessed different cell behaviors, for example, cell attachment, cell survival and cell spreading during cell-matrix interactions. We set up a two-photon microfabrication platform for a precise and spatial control of the biochemical and the mechanical properties independently in a 3D protein-based biomimetic cell niche. We also established a single-cell based cell niche platform by using this technology. Our further studies will focus on incorporating more bioactive signals to better mimic the native 3D cell niche microenvironment.-
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.lcshExtracellular matrix proteins-
dc.subject.lcshMicrofabrication-
dc.titleMultiphoton crosslinking-based protein micropatterning : a platform to study interactions between cells and matrix niche-
dc.typePG_Thesis-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineMechanical Engineering-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_991044019381603414-
dc.date.hkucongregation2018-
dc.identifier.mmsid991044019381603414-

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