Bioprinting spatially guided functional 3D neural circuits with agarose-xanthan gum copolymer hydrogels

Abstract

Engineered three-dimensional (3D) tissue models are being used as predictive human in vitro assays for drug discovery and development. Tissue engineering technologies such as bioprinting are now available which use mixtures of polymeric hydrogels and cells for the construction of biomimetic engineered 3D tissue models. Many of the polymeric hydrogels used for bioprinting require post-printing processing steps which might hinder their application directly in multi-well plate platforms, thus limiting their utility in a drug screening setting. Here we describe an agarose and xanthan gum copolymer hydrogel (AG-XG) that has optimal rheological properties for high shape fidelity with extrusion-based printing, has long term stability in cell culture conditions, and is "ready-to-use" after printing, not requiring post-printing processing treatments, making it ideal for applications in multi-well plate format. This AG-XG hydrogel is non-degradable and has non-cell permissive features which makes it ideal to create customized spatially guided cellular patterns to enhance relevant tissue geometry and function. As a proof-of-concept, we show that a bioprinted AG-XG hydrogel casting mold significantly enhances functional connectivity of an engineered 3D neural circuit model made using human iPSC-derived GABAergic and dopaminergic neurons and astrocytes. The bioprinted AG-XG mold promotes the formation of strong functional synaptic connections between two spatially separated neuronal regions, as measured with calcium and optogenetic-based fluorescent biosensors with a customized fiber photometry device. The high shape fidelity of the AG-XG hydrogels described here enables the biofabrication of precisely positioned and spatially designed cellular models, in muti well-based platforms used for drug screening. The process of printing these AG-XG hydrogels uses commercially available extrusion-based bioprinters and can therefore be easily implemented in translational laboratories doing tissue modeling and drug screening without the need of additional specialized bioengineering equipment.

Keywords: 3D cellular patterning; Agarose; Copolymeric hydrogel; Drug screening; Extrusion-based bioprinting; Multi-well plate format; Neural circuits; Tissue engineering; Tissue models; Xanthan gum.