Solid-supported lipid bilayer as a platform to study cell-extracellular matrix interactions

• Main Author: Vafaei, Setareh
• Other Authors: Cho Nam-Joon
• Format: Theses and Dissertations
• Language: English
• Published: 2017
• Subjects: DRNTU::Engineering::Materials::Biomaterials
• Online Access: http://hdl.handle.net/10356/72888
• Institution: Nanyang Technological University

Development of functionalized surfaces that can interface with cells is essential for many biotechnological applications ranging from routine cell culture to advanced applications such as cell-based diagnostics, tissue engineering, medical implants and biosensor development. Supported lipid bilayer (SLB), is a versatile synthetic platform which mimics the fundamental properties of native biological membranes and enables studying membrane-associated cellular processes in sufficiently simplified and more controlled conditions. SLBs composed of zwitterionic lipids are resistant to nonspecific adsorption of cells and proteins. However, they can be functionalized with specific cell recognition motifs by introducing lipids with chemically selective headgroups into the bilayer for direct covalent or non-covalent coupling of biomolecules. The primary aim of this thesis work has been to explore and advance the application of SLBs as cell-surface mimetic substrates for cell interaction studies, with the special focus on functionalization of SLBs with the extracellular matrix (ECM) components. The ECM is a tissue-specific network of extracellular bio-active molecules that provides support and anchorage for cells and plays the key role in numerous cellular processes including cell growth and differentiation as well as intercellular communications. The behaviour of cells in a tissue is regulated by both chemical as well as mechanical signals arising from their microenvironment. First, we developed an experimental platform using SLB functionalized with ECM proteins for mimicking a range of substrate rigidities by tuning the bilayer viscosity. We used a recently developed method of SLB formation called solvent-assisted lipid bilayer (SALB) formation to fabricate bilayers containing various fractions of a functional lipid with a reactive headgroup to attach two major ECM components; collagen type I (Col I) and fibronectin (FN) covalently to the bilayer surface. Using, quartz crystal microbalance with dissipation (QCM-D), we demonstrated that the density of the proteins can be controlled by the fraction of reactive lipids included in the lipid precursor solution used in the SALB method. The ECM-SLB platform, supported the cell attachment, growth, and function. Importantly, fluorescent recovery after photobleaching (FRAP) analysis showed that bilayer retained its fluidity after protein conjugation and cell adhesion. The fluid nature of the bilayer led to the lateral displacement of anchored Col and FN and their accumulation underneath the cell in response to the traction forces exerted by the cells during the cell spreading process. We could control the displacement by increasing the viscosity of the membrane via incorporation of a high fraction of cholesterol (Chol) (up to 40%), a possibility offered by the SALB method. Second, we investigate the biological activity of Col I and FN which are covalently attached to the SLB and compared with those of nonspecifically adsorbed to SiO2 surface. The characterization of protein adsorption by QCM-D revealed that Col I and FN that attached to SLB have higher structural flexibility than those adsorbed onto SiO2, and their activity was higher with respect to Col I-FN interaction and cell adhesion efficiency. Finally, in attempt to develop a substrate which more closely mimics the native cell-surface environment, we functionalized the SLB with decellularized extracellular matrix (dECM) components. The dECM was obtained through a combination of chemical and enzymatic treatments of mouse adipose tissue and was solubilized under acidic conditions. We demonstrated that dECM functionalized bilayers sustain cell growth and maintain their function. The strategy and results presented in this thesis work stress the potential of ECM-SLB platform for studying cell-matrix interactions as well as studying cellular processes that occur at the membrane interface.