[en] Cell culture models in 3D have become an essential tool for the implementation of cellular models of neurodegenerative diseases. Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons from the substantia nigra. The study of PD at the cellular level requires a cellular model that recapitulates the complexity of those neurons affected in PD. Induced Pluripotent Stem Cells (iPSC) technology is an efficient method for the derivation of dopaminergic neurons from human neuroepithelial stem cells (hNESC), hence proving to be a suitable tool to develop cellular models of PD. To obtain DA neurons from hNESC in a 3D culture, a protocol based on the use of small molecules and growth factors was implemented in a microfluidic device (OrganoPlate). This non PDMS device is based on the use of phaseguide (capillary pressure barriers that guide the liquid air interface) technology and the hydrogel matrigel as an extra cellular matrix surrogate. To characterize the morphological features and the electrophysiological activity of wild type hNESCs differentiated neuronal population, with those differentiated neurons carrying the LRRK2 mutation G2019S, a calcium imaging assay based on the use of a calcium sensitive dye (Fluo-4) and image analysis methods, were implemented. Additionally, several aspects of fluid flow dynamics, rheological properties of matrigel and its use as surrogate extracellular matrix were investigated. Final characterization of the differentiated neuronal population was done using an immunostaining assay and microscopy techniques. The yields of differentiated dopaminergic neurons in the 2 lane OrganoPlate were in the range of 13% to 27%. Morphological (length of processes) and electrophysiological (firing patterns) characteristics of wild type differentiated neurons and those carrying the LRRK2 mutation G2019S, were determined applying an image analysis pipeline. Velocity profiles and shear stress of fluorescent beads in matrigel flowing in culture lanes of the 2 lane OrganoPlate, were estimated using particle image velocimetry techniques. In this thesis, we integrate two new technologies to establish a new in vitro 3D cell based model to study several aspects of PD at the cellular level, aiming to establish a microfluidic cell culture experimental platform to study PD, using a systems biology approach.
Luxembourg Centre for Systems Biomedicine (LCSB): Systems Biochemistry (Fleming Group)