Development of 3D Hydrogel-Based In Vitro Circuits with Cocultures of Human Sensory Neurons and Schwann Cells

Engineered <i>in vitro </i>neural networks are promising platforms to rapidly screen drugs and study information flow in the nervous system [1]. While existing polydimethylsiloxane (PDMS)-based microfluidic platforms offer precise architecture and connectivity, the cultured neurons grow inside microchannels on a planar multielectrode array (MEA) substrate in a two-dimensional fashion [2]. To better mimic the native extracellular matrix (ECM) microenvironment, 3D hydrogel scaffolds can be designed so that encapsulated cells can be expected to exhibit more physiological behavior [3].<br/>Here, we propose a hybrid approach by filling the PDMS microstructure with hydrogels to offer both a controlled topology and a physiologically relevant microenvironment to the neuronal culture. First, a gelatin methacryloyl (GelMA) hydrogel was engineered by incorporating ECM components and tuning its mechanical properties to match the native niche environment. The hydrogel material was able to 1) support the growth of human iPSC-derived sensory neurons for more than 9 weeks 2) support co-culture with human Schwann cells where the latter could align along the axons. Next, microchannels were filled with the optimized hydrogel precursor, and after crosslinking, the neurons and glial cells were seeded as pre-aggregated spheroids in desired location(s). This enabled topologically defined growth of neurites in 3D.<br/>By further tuning the hydrogel type and its physico-mechanical properties, the microstructures could be filled and tailored for specific cell types, while the underlying MEA allows for the recording of neuronal activity over time. This platform could offer a promising tool to study cell-cell interactions of single cultures or co-cultures, and to efficiently test the potential of various drugs in a more translational manner.<br/><br/>[1]. Aebersold, M. J., Dermutz, H., Forro, C., Weydert, S., Thompson-Steckel, G., Vörös, J., & Demko, L. (2016). “Brains on a chip”: Towards engineered neural networks. Trends in Analytical Chemistry, 78, 60-69.<br/>[2]. Forró, C., Thompson-Steckel, G., Weaver, S., Weydert, S., Ihle, S., Dermutz, H., Aebersold, M. J., Pilz, R., Demkó, L., & Vörös, J. (2018). Modular microstructure design to build neuronal networks of defined functional connectivity. Biosensors and Bioelectronics, 122, 75-87.<br/>[3] Huh, D., Hamilton, G. A., Ingber, D., E. (2011). From 3D cell culture to organs-on-chip. Trends in Cell Biology, 21, 12, 745-754.