| Reference : Automated optimisation of stem cell-derived neuronal cell culture in three dimensiona... |
| Dissertations and theses : Doctoral thesis | |||
| Engineering, computing & technology : Multidisciplinary, general & others | |||
| Systems Biomedicine | |||
| http://hdl.handle.net/10993/39870 | |||
| Automated optimisation of stem cell-derived neuronal cell culture in three dimensional microfluidic device | |
| English | |
| [fr] Optimisation automatisée de la culture de cellules neuronales dérivées de cellules souches dans un dispositif microfluidique tridimensionnel | |
Kane, Khalid  [University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > >] | |
| 20-Jun-2019 | |
| University of Luxembourg, Luxembourg | |
| DOCTEUR DE L’UNIVERSITÉ DU LUXEMBOURG EN SCIENCES DE L’INGENIEUR | |
| 220 | |
| Schwamborn, Jens Christian | |
| Wagner, Christian | |
| Krüger, Rejko | |
| Zilian, Andreas | |
| Wixforth, Achim | |
| [en] Robotics ; Automation ; Rheology ; Matrigel ; hydrogel ; microfluidic ; Neuronal cell culture | |
| [en] This dissertation is a compilation of publications and manuscripts that aim 1) to integrate an automated platform optimised for long term in vitro cell culture maintenance for Parkinson’s disease, long term live cell imaging and the handling of many cell lines, 2) to combine physics principles with imaging techniques to optimise the seeding of Matrigel embedded human neuroepithelial stem cells into a three-dimensional microfluidic device, and 3) to combine engineering principles with cell biology to optimise the design of a three-dimensional microfluidic system based on phaseguide technology.
In the first publication manuscript, we investigated Matrigel as a surrogate extracellular matrix in three-dimensional cell culture systems, including microfluidic cell culture. The study aimed at understanding and characterising the properties of Matrigel. Using classical rheological measurements of Matrigel (viscosity versus shear rate) in combination with fluorescence microscopy and fluorescent beads for particle image velocimetry measurements (velocity profiles), the shear rates experienced by cells in a microfluidic device for three-dimensional cell culture was characterised. We discussed how the result of which helped to mechanically optimise the use of Matrigel in microfluidic systems to minimise the shear stress experienced by cells during seeding in a microchannel. The second manuscript proposes a methodology to passively control the flow of media in a three-dimensional microfluidic channel. We used the fluid dynamic concept of similitude to dynamically replicate cerebral blood flow in a rectangular cross-sectional microchannel. This similarity model of a target cell type and a simple fluid flow mathematical prediction model was used to iterate the most optimum dimensions within some manufacturing constraints to adapt the design of the OrganoPlate, a cell culture plate fully compatible with laboratory automation, which allowed its re-dimension to achieve over 24h of flow for the culture of human neuroepithelial stem cells into midbrain specific dopaminergic neurons. In the third publication manuscript, we propose an automated cell culture platform optimised for long-term maintenance and monitoring of different cells in three-dimensional microfluidic cell culture devices. The system uses Standard in Laboratory Automation or SiLA, an open source standardisation which allows rapid software integration of laboratory automation hardware. The automation platform can be flexibly adapted to various experimental protocols and features time-lapse imaging microscopy for quality control and electrophysiology monitoring to assess cellular activity. It was biologically validated by differentiating Parkinson’s disease patient derived human neuroepithelial stem cells into midbrain specific dopaminergic neurons. This system is the first example of an automated Organ-on-a-Chip culture and has the potential to enable a versatile array of in vitro experiments for patient-specific disease modelling. Finally, the fourth manuscript initiates the assessment of the neuronal activity of induced pluripotent stem cell derived neurons from Parkinson’s Disease patients with LRRK2-G2019S mutations and isogenic controls. A novel image analysis pipeline that combined semi-automated neuronal segmentation and quantification of calcium transient properties was developed and used to analyse neuronal firing activity. It was found that LRRK2-G2019S mutants have shortened inter-spike intervals and an increased rate of spontaneous calcium transient induction than control cell lines. | |
| Professionals | |
| http://hdl.handle.net/10993/39870 | |
| H2020 ; 668738 - SysMedPD - Systems Medicine of Mitochondrial Parkinson’s Disease |
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