Biomimetic coatings on technical substrates for cell culture

Svetlova, Anastasia; Offenhäusser, Andreas (Thesis advisor); Pich, Andrij (Thesis advisor)

Aachen : RWTH Aachen University (2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022

Abstract

Ability to study living cells by a non-invasive methods, such as cell culture, was a foundation for modern biological and biotechnological advances. As it was discovered that a cell function cannot be separated from its surroundings, a quest to recreate living tissues in controlled laboratory conditions began. The researchers are constantly trying to impove methodology to study cells in environments that resemble natural tissue environments the most. The creation of complex environments requires precise controll of properties of the underlying substrate. The project consisted of the development of the coating that mimics a natural cell membrane and is a suitable platform for cell culture, and the adaptation of the electrical measurement system. Solid-supported lipid bilayers (SLBs) were used as the basis for the biomimetic coatings, and graphene field-effect transistors (GFETs) as the measurement system. The major challenge of the first part of the project was to overcome hydrophobicity of graphene surface, as lipid bilayers are not formed on the pristine graphene. An electrochemical oxidation was tested as a route to overcome that. However, it was revealed that chemical vapor deposited (CVD) graphene is incompatible with this method, as the process, instead of the uniform functionalization, destructed the layer integrity. Moving along with the theme of a link between electrochemical properties and the surface quality, the detailed research on the origins of the leakage currents of graphene electrolyte-gated transistors was performed. It was determined that value can be used to evaluate and monitor surface properties of graphene in situ. Results of these findings were summarized in publications [1, 2].To produce biomimetic coatings, extracts from cell membranes of HEK293 cells were made by the chemical vesiculation method. Similarity of the structural backbone of cell vesicles and SLBs made from artificial lipid mixes allows them to fuse into composite layers that are enriched by cell membrane molecules, such as glycolipids and proteins, that are important for cell-cell interactions. The chemical vesiculation process was studied with the flow cytometry and the fluorescent imaging methods, and fusion of layers - by the quartz crystal microbalance in a microfluidic system. Two cell culture models were used to characterize their interactions with the biomimetic layers: HL-1, the cardiomyocyte-like cell line, and primary cortical neurons from rat embryo brains. These cells have distinctly different properties and demands to their environment, so different characterization pathways were selected for each. For HL-1 cells, which are, essentially, muscle cells, their cytoskeleton was monitored. The ability to form a developed cytoskeleton that supports the cell shape and contractions depends on the strength of their interaction with the substrate and its softness. Biomimetic coatings modified with different amounts of cell extracts allowed to tune the optimal conditions for cell adhesion. These findings were published in [3].For neuronal cells in vitro the development of the cell polarity is their main property. The neurite elongation dynamics on different coatings were analyzed and it was discovered that interactions of neurons with highly fluid SLB-based coatings have unique features during the earliest hours of the development.