Electrophysiology of human induced pluripotent stem cell-derived neurons cultivated on micro- and nanostructured substrates

Abstract

Merging human induced pluripotent stem cell (iPSC)-derived neurons with the advantages of functionalized micro- and nanostructured cell culture substrates might open up new pathways for, e.g., bioengineering and regenerative medicine. This advanced type of cells, however, is exceptionally difficult to cultivate making the feasibility of such a unique combination uncertain.

In this thesis, the cultivation of human iPSC-derived neurons was thus investigated on a selection of micro- and nanostructured substrates to identify potential challenges such as an altered differentiation outcome of the Petri dish-established culturing protocols due to the modified growth environment. Specifically, the neuronal differentiation while being cultured on nanowire (NW) arrays as well as the formation of neuronal networks inside 3D-printed microscaffolds were tested and verified. Both types of substrates are of keen interest in stem cell culture: on the one hand, NW arrays are able to facilitate, for example, the delivery of biological payloads, or to enhance the electrochemical coupling to the substrate for superior sensing/stimulation. On the other hand, 3D-printed microscaffolds allow for establishing defined neuronal networks and improved resemblance of the three-dimensional nature of the human brain for brain-on-a-chip (BoC) applications. The functionality of the neurons, i.e., the quality of the generated cells, and the neuronal networks were analyzed using electrophysiological patch clamp measurements. Immunocytochemistry (ICC) served to substantiate the neuronal characterization. The interactions of the cells with the substrates were analyzed using confocal laser scanning microscopy (CLSM) and cross-sectional scanning electron microscopy (SEM) prepared by focused ion beam (FIB) milling. One key result of this work is the demonstration of equal neuronal differentiation on several types of NW arrays featuring substantially diverse substrate geometries such as altered NW lengths, array pitches, and NW diameters compared to planar controls. The positive outcome of the extensive study suggests that prospectively, the well-established applications of NW arrays, which have so far only been demonstrated on basic cell lines and primary cells from animals, might be applicable to human iPSC-derived neurons. Another major achievement of this thesis is the generation of defined neuronal networks inside the 3D-printed microscaffolds in which the human iPSC-derived neurons showed spontaneous excitatory postsynaptic currents (sEPSCs) indicating functional network activity. Here, the versatility of the two-photon polymerization (2PP) direct laser writing (DLW) technique employed to prepare the 3D scaffolds enables the fabrication of scaffolds with arbitrarily complex network topologies which might then enhance, for instance, brain organoid cultivation and BoC applications.

Concludingly, both types of the utilized hybrid systems might in the future set new standards in life sciences and biotechnology by facilitating unconventional approaches for, e.g., personalized high-throughput drug screenings and neurodegenerative disease studies.