Hebel, Diana

The investigation of the response of the immune system, which is influenced by the communication between cells of the same as well as different species (interkingdom signalling), is a crucial part in biomedical research. Especially the sensing of signalling molecules that are produced by bacteria can induce a response of eukaryotic cells present in the immune system. For the studies of cellular response in vitro, the extracellular matrix (ECM), which surrounds the cells in their natural environment, needs to be simulated. The creation of an artificial three-dimensional (3D) environment is essential and affects the cell adhesion, proliferation as well as migration. Thereby, the cell detachment and separation can be induced. In this Thesis, different approaches to generate suitable cell culture platforms were explored and their suitability for future work in cell communication investigated. In particular, polymer brushes as well as lipid bilayers were examined as approaches to spatially and temporally control selective cell attachment and detachment. Furthermore, hydrogel-based scaffolds were prepared and investigated regarding their suitability to create viable environments for cell encapsulation. Thermoresponsive poly(di(ethylene glycol) methyl ether methacrylate) (PDEGMA) brushes were successfully used for selective separation of human umbilical vein endothelial cells (HUVEC) from a coculture with macrophages without the use of additional compounds, such as releasing agents or antibodies. The decrease of the temperature below the transition temperature of 35 °C for (5 ± 1) nm thin PDEGMA brushes, resulted in a change of the chain conformation and thus cause a desorption of cell adhesion proteins. HUVECs and macrophages attached and spread in a coculture on (5 ± 1) nm thin PDEGMA brushes at 37 °C. In contrast to macrophages HUVECs, which possess a lower adhesion strength, detached from the brushˈ surface after decreasing the temperature to 22 °C. HUVECs could be reseeded on a new surface with a yield of 71 % and a purity of almost 100 %. Square-shaped, patterned polymeric lipid bilayers that were modified with PDEGMA and poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) brushes with brush thicknesses of 26 nm and 20 nm, respectively, were investigated in terms of selective cell attachment. Mouse fibroblasts (NIH 3T3) cells were able to attach and spread on polymeric lipid bilayer. In contrast, polymer brushes possess cell repulsive properties, so that the cells are only able to attach on bare glass squares. The use of patterned polymeric lipid bilayers, which are modified with polymer brushes, allows a selective cell attachment. The adhesion and spreading of cells inside different hydrogels with various geometries were investigated. The encapsulation of NIH 3T3 cells and pancreatic tumor cells (PaTu 8988t) inside chitosan microbeads cross-linked with glycerol phosphate disodium salt (CS + GP) as well as inside alginate — fibrin microbeads were not successful due to the too harsh conditions of the gelling bath and the lack of cell adhesion properties, respectively. However, a hydrogel consisting of alginate dialdehyde (ADA) with a degree of oxidation of 30 % and gelatine modified with carbohydrazide (GelCHD) in a ratio of 1 : 3 were shown to be useful, afford attachment, spreading, and proliferation of encapsulated cells. Finally, the response of encapsulated NIH 3T3 cells, in the above-mentioned hydrogel, to the signalling molecule N-(3-oxododecanoyl)-L-homoserine lactone (HSL) was examined. A pronounced decrease of the cellsˈ viability from 90 % to 71 % and an increase in the intracellular Ca2+ concentration was detected after treatment with HSL, confirming a response of the cells on the signal molecules. Overall, the results reported in this Thesis highlight the potential of polymer brushes, lipid bilayers as well as hydrogel scaffolds for the encapsulation of cells and the investigation of cellular communication processes.

The authors report on a mild, label‐free, and fast method for the separation of human umbilical vein endothelial cells (HUVEC), which are relevant cells, whose use is not limited to studies of endothelial dysfunction, from cocultures with macrophages to afford HUVEC in ≈100% purity. Poly(di(ethylene glycol)methyl ether methacrylate) (PDEGMA) brushes with a dry thickness of (5 ± 1) nm afford the highly effective one‐step separation by selective HUVEC detachment, which is based on the brushes' thermoresponsive behavior. Below the thermal transition at 32 °C the brushes swells and desorbs attached proteins, resulting in markedly decreased cell adhesion. Specifically, HUVEC and macrophages, which are differentiated from THP‐1 monocytes, are seeded and attached to PDEGMA brushes at 37°C. After decreasing the temperature to 22°C, HUVEC shows a decrease in their cell area, while the macrophages are not markedly affected by the temperature change. After mild flushing with a cell culture medium, the HUVEC can be released from the surface and reseeded again with ≈100% purity on a new surface. With this selective cell separation and removal method, it is possible to separate and thereby purify HUVEC from macrophages without the use of any releasing reagent or expensive labels, such as antibodies.

Hebel, Diana

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