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Spatio-temporal control of neuronal network formation with micropatterned thermoresponsive cell culture substrates

Laura V.J. Behm1, 2*, Susanna Gerike1, 3, Felix Pfisterer1, Philipp Wysotzki4, Katja Uhlig1, Werner Baumann4, Frank Bier1, 5, Claus Duschl1 and Michael Kirschbaum1
  • 1 Fraunhofer-Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam-Golm, Molecular and Cellular Bioanalytics, Germany
  • 2 Berlin School for Regenerative Therapies, Charité University Hospital, Germany
  • 3 Freie Universität Berlin, Germany
  • 4 University of Rostock, Chair of Biophysics, Germany
  • 5 University of Potsdam, Institute of Biochemistry and Biology, Germany

In vitro neuronal networks with defined connectivity are an excellent tool to investigate network properties such as pre- and postsynaptic mechanisms of neuronal cells. This becomes increasingly important with the emerging possibilities of human induced neurons as an alternative to rodent cells for example for in vitro disease modelling. However, most protocols for the formation of in vitro neuronal networks do not provide sufficient control of the direction and timing of neuronal connections with simultaneous access for common analysis tools such as immunocytochemistry or electrophysiological recordings. We have developed micropatterned thermoresponsive cell culture substrates for temporally and spatially controlling the formation of simplified neuronal circuits in vitro. For our approach we used thermoresponsive polymer (TRP) coatings that can be switched during cultivation from a cell-repellent to a cell-permissive state by switching the temperature of the substrate e.g. from 33°C to 37°C. Key elements of the TRP-coated substrates are TRP-free patterns in the form of 50 µm wide somal adhesion spots with 200 µm distance that are interconnected by TRP-free neurite pathways of 5 µm width and 150 µm length. Since the neurite pathways are a bit shorter than the distance between two somal adhesion spots, a 50 µm wide gap of TRP is created between neurite pathways and adhesion spots which allows us to control neuronal cell growth and neurite outgrowth between these two structures. Neuronal cells were initially confined to the TRP-free pattern by forcing the surrounding TRP to its cell-repellent state. We kept small groups of human neuronal SH-SY5Y cells at 33°C within these patterns for 4-5 days. Upon switching the TRP to the permissive state by a temperature increase to 37°C the cells extended their neurites across the gaps of TRP towards neighbouring cells. This allowed us to control the formation and connectivity of small simplified neuronal networks in time and space. Immunocytochemistry on these artificial neuronal networks showed that the neuronal marker ß-tubulin III was expressed as in differentiated cells on standard surfaces, indicating that the cells keep their differentiated neuronal phenotype. We currently transfer our approach to primary neuronal cells. Another important next step will be local heating on the microscale for an improved spatial control over the dynamic TRP surface.

Acknowledgements

We acknowledge financial support by the German Research Foundation DFG (DU 167/4-1) and the Berlin-Brandenburg School for Regenerative Therapies GSC 203.

Keywords: in vitro Neuronal Networks, micropatterning, thermoresponsive cell culture substrate, spatiotemporal guidance, Neurite outgrowth, SH-SY5Y cells

Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.

Presentation Type: Oral Presentation

Topic: Microphysiological systems

Citation: Behm L, Gerike S, Pfisterer F, Wysotzki P, Uhlig K, Baumann W, Bier F, Duschl C and Kirschbaum M (2019). Spatio-temporal control of neuronal network formation with micropatterned thermoresponsive cell culture substrates. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00037

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Received: 14 Mar 2018; Published Online: 17 Jan 2019.

* Correspondence: Ms. Laura V.J. Behm, Fraunhofer-Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam-Golm, Molecular and Cellular Bioanalytics, Potsdam, 14476, Germany, laura.behm@izi-bb.fraunhofer.de