Real-time multi-parametric monitoring of neural stem cell differentiation using on-line culture system

Cell replacement therapy (CRT) is a promising restorative approach proposed for treatment of neurodegenerative diseases e.g., Parkinson’s disease (PD) characterized by loss of dopaminergic (DA) neurons. This approach aims to replace the injured or dead neurons and has the potential for treatment of PD patients. In order to initiate clinical trials neurons suitable for implantation have to be generated from stem cells in culture that provides a sustainable source for CRT. Neural stem cells (NSCs) has a potential in cell replacement strategies, however prior to consider implantation of NSCs it is crucial to characterize their phenotype and functional DA properties.
The aim of my project was to adapt the existing EXCELL microfluidic platform to characterize cellular dynamics with non-invasive multi-parameter detection during NSC differentiation into DA neurons.
For the purpose of characterization of the NSC differentiation process relevant techniques has to be implemented in the EXCELL platform. Hence, we investigated several aspects of different non-invasive real-time analysis techniques.
The research reported in this thesis includes following techniques:
1) Electrochemical impedance spectroscopy (EIS) applied for in-depth analysis of NSC differentiation (Paper I and Paper II).
2) A combination of amperometry, EIS monitoring and microscopic visualization in microfluidics assays to monitor a specific cell population over time (Paper III).
3) Molecular beacon bio-imaging to track real-time changes of specific DA gene expression pattern.
4) Calcium influx imaging to follow the functionality of DA neurons.
We showed, that most individual techniques tested were feasible and optimized protocols could provide the basis for further implementation to the platform system.
This Ph.D. project was an extension of the EU-funded FP7 project EXCELL (Exploring Cellular Dynamics at Nanoscale) with the overall aim to construct innovative tools for investigation of real-time cellular dynamics. The main focus of the EXCELL project was to construct a multi-parameter microfluidic cell culture and detection platform compatible with optical and electrochemical techniques to monitor cellular changes non-invasively.