In vitro investigation of neuronal polarization and growth in a microstructured environment
In vitro onderzoek naar neuronale polarisatie en groei in een microgestructureerde omgeving
Micholt, Liesbeth; S0163301
To ultimately restore function to damaged nerves after injuries or diseases, we need to understand better how neurons respond to changes in their immediate surroundings. This micro-environment comprises cues of different nature; biophysical elements like micro-topographies and electrical events, but also surface-bound and soluble chemical gradients. Studying aspects of the microenvironment separately and in concert in a well-controlled in vitro situation allows to gain valuable insight on the fundamental mechanisms governing cellular responses.The aim of this work was to investigate how different micro-engineered environments modulate the cellular polarization, growth and response. Inthe first part we studied how neurons polarize in response to topographic stimuli and how this affects the outgrowth behavior. To this end we micro-fabricated surfaces having particular, highly reproducible, topographic features, and demonstrated their ability to influence the early phases of neuronal polarization, notably the formation of the first neurite, axonal differentiation and growth. In the second part we refined the cellular micro-environment by exposing growing neurons simultaneously to distinct guidance cues. For this purpose we designed a novel integrated micro-fabricated device combining soluble gradients with topographic surface features. We examined the responses of neurites extending from neurons cultured on substrates of different local micro-topographies and geometric organizations, combined with attractive and repulsive soluble chemicalcues. The results show the potential for powerful synergies between biophysical and chemical guidance cues and are relevant to understand complex guidance control in vivo during development and regeneration.On top, these new insights aid in the creation of model neuronal networks interfaced with artificial growth substrates and in drug screening applications. In the third part of this thesis we focused on interfacing electrogenic cells with subcellular scale electrodes. Achieving single-cell resolution during electrical stimulation can help to gain knowledge on the effect of modifications in an individual cell on an entire network. Here, we showed that electric stimulation pulses can be delivered accurately to different cell types, and there are analogous features in their response to stimulation. In HL-1 cells we found that the Ca2+ signals can have different types of activation phenotypes and we presented a model to describe the effects in a more generalized fashion. Neurons react to stimulation in a dose-response manner. Future research has to clarifyif the activation phenotypes can also be found in neurons, and if in HL-1 cells the dose-response reaction is conserved.This thesis work demonstrates the great potential that lies in the combinatorial application of different biophysical and chemical cues to controllably modify the immediate cellular micro-environment. Future applications are situated in test platforms useful for the fundamental, in vitro study of cell response tocombined topographic, chemical and electrical stimuli. Ultimately, the lessons drawn from these studies can support the development of a multifunctional, tissue-engineered implant leading to improved regeneration after neuronal lesions and generating treatments not previously possible, and can help to gain new insights in fundamental disease mechanisms.