Development of novel hydrogel substrates for neuronal 3-D cultures

Kurzfassung

For over 50 years cell cultures are commonly used to do basic and advanced research on cells derived from organisms to understand their function and to test new potential applications. In order to investigate different isolated cell types, especially from mammals, and to test their behavior to different stimuli or chemicals, scientists had established the so called “2D” cell cultures on various surfaces like glass or plastics, which can also be coated with adhesion-promoting substrates, e.g. amino acids or collagen to enhance cell growth and survival. With this well-established 2D cell culture it is possible to observe differentiation, growth behavior, cellular adaptations and gene expression changes to different stimuli unlike to the situation in vivo. In comparison to cells in their native tissue environment the results from experiments in 2D cultures are similar but might differ in certain aspects and have to be critically considered. To avoid this problem a new form of cell culture was invented: 3D cultures. New discoveries in the field of biochemistry, allow to seed cells on and in different scaffolds like polysaccharide and protein-based hydrogels, which are designed to mimic the tissue of origin to cultured cells as physiologically as possible. Therefore, the aim of this thesis is to screen different hydrogels consisting of polysaccharides and silica derivates for their suitability to support the growth, survival and network building of primary neuronal cells, i.e. astrocytes. Astrocytes are localized in the brain and support neurons by taking on various tasks like the active control of neuronal activity and synaptic neurotransmission by regulation of e.g. calcium homeostasis. Novel artificial 3D scaffold for further investigations on the interaction between primary astrocytes and neurons were established. Further, the suitability of 3D cultivation for biomedical applications, such as inhibiting glial scar formation was investigated. Several new established synthesis protocols for organic, inorganic and functionalized hydrogels were tested. Furthermore, protocols for immunofluorescence, Western Blots and analytical methods like the dynamic mechanical analysis or scanning electron microscopy to determine the stiffness and pore sizes in comparison to brain tissue were adapted. It could be shown that most of the tested hydrogels are biocompatible but have a lack of sufficient cell adhesion sites on their basic constituents. To overcome this problem, two gel types were functionalized with well-known peptides, which showed improved cell growth after several days. Beside this, certain gel types depicted a similar stiffness compared to murine brain and ranked in their pore sizes of a few nanometers, as is also found in neuronal ECM, to several micrometers. Comparing 3D to standard 2D cultured cells, all successful 3D cultured astrocytes showed different morphological features with a tendency for astrocytes in certain gel types to develop similar to the in vivo morphology.