Benjamin Simona, Ectica Technologies AG, Zurich, Switzerland
Vincent Milleret, Greta Thompson-Steckel, Laboratory of Biosensors and Bioelectronics, ETH Zurich, Zurich, Switzerland

Hydrogels are widely used as an artificial extracellular matrix to grow neural cells in a three-dimensional (3D) environment. 3D cultures have the advantage of closely recapitulating aspects of the human tissues including the architecture and organization among cells (cell-cell and cell-matrix interactions) and more physiologically relevant diffusion characteristics. In 2014, Kim and co-workers reported in Nature a human neuronal cell culture 3D model in Matrigel [1]. The authors demonstrated that the 3D cell culture conditions promoted neural maturation compared to 2D and observed a dramatic increase in the levels of 4-repeat adult Tau isorforms, which are essential for reconstituting tauopathy in vitro. Finally, the authors demonstrated the presence of insoluble extracellular amyloid β deposits, an aspect of the disease that is not recapitulated in conventional 2D cultures because secreted Aβ freely diffuses in a large volume of media and is not confined in a matrix surrounding the cells. Overall, adopting 3D neuronal cultures to establish Alzheimer’s disease in vitro models is a promising strategy to recapitulate the biological relevance of in vivo model with the advantage of using cells of human origin.

Culturing cells within hydrogels normally requires their encapsulation in the hydrogel during its formation [2]. The encapsulation process is required because of the very limited penetration in 3D of cells deposited on the surface of a pre-assembled gel. The encapsulation process reduces the throughput of the assay, increases the variability and necessitates the simultaneous seeding of different cell populations in case a co-culture assay is envisaged. To tackle these important limitations in the use of hydrogels for 3D cell culture, we developed hydrogels with an “in-depth surface density gradient” promoting the infiltration in 3D of cells deposited on the hydrogel surface (3DProSeedTM hydrogels) [3]. Here we report a preliminary study of the culture of iPSC-derived neuronal cells in 3DProSeed, with the long term goal of establishing automation-compatible 3D models for neurodegenerative and neurotoxicology screenings while maintaining the highest level of high-content screening (HCS) workflow integration and automation-compatibility. Neurons seeded on 3DProSeedTM hydrogels rapidly extend the neurites in the 3D gels and are viable for at least 19 days. Preliminary functional assays of the calcium activity demonstrates the formation of functional neuronal networks in 3D. Thanks to the presence of the hydrogel surface gradient it is possible to add different cell populations at different time points. Thus, iPSC-derived astrocytes were sequentially seeded and after only 5 days, astrocytes penetrated the hydrogel in 3D and were found associated with the neurites. In conclusion, we propose a novel hydrogel platform for the development of assays in neurodegeneration and neurotoxicology in 3D, providing simplicity in use, the highest level of automation compatibility and the enabling power of the sequential seeding for establishment of co-culture systems.

[1] Se Hoon Choi, Young Hye Kim, et al. , Nature 515, 274–278 , 2014.
[2] Steven R Caliari & Jason A Burdick, Nature Methods 13, 405–414 , 2016
[3] MIlleret V., Simona B.R. et al. Healthc. Mater. 2014 & PCT/EP2012/076426 [4] Zhang, Y. et al., Neuron 78, 785-798, 2013