Fabrication of Designable and Suspended Microfibres via Low Voltage Electrospinning Patterning towards Replicating Extracellular Matrix Cues for Tissue Assembly

Abstract

Lab-grown tissues have tremendous potential to accelerate drug discovery and identify some of the underlying mechanisms behind diseases. The native extracellular matrix (ECM) of tissues is a complex, hierarchical fibrous protein structure with delicate mechanical properties that guides tissue assembly and regeneration. Existing biomaterial fabrication techniques struggle to simultaneously attain: micro/nano-scale fibril feature resolution, low bulk stiffness and the 3D organisation crucially provided by the ECM without comprising cell motility. This work utilises 3D printing and low voltage electrospinning patterning synergistically to address these conflicting engineering challenges and act as a minimalist guide for 3D cell growth. A version of low voltage electrospinning patterning was adapted as a sequential process on a modified 3D printer. Applied voltage and 3D printed geometry can modulate the suspended behaviour of electrospun fibres that span between 3D printed support pillars, a parametric study characterised threshold conditions and established a predictive model for patterning suspended fibres. The accuracy with which suspended fibres followed the in-plane tool path was also assessed. Scanning Electron Microscopy imaging measured fibre diameters 1-5 μm and mechanical testing examines the properties for a given layer of dry fibres. The configuration demonstrated unique patterning of stacked suspended fibre layers in multiple orientations. Tissue scaffolding applications were explored in 2D and 3D. In 2D, gelatin fibres were patterned as a topographic cue to direct mesenchymal stem cells towards the osteogenic lineage. For 3D cell culture, the use of suspended fibre devices was investigated to improve the efficiency of cerebral organoid assembly. Pursuing these applications led to further refinement of the fibre fabrication technique and the development of targeted cell seeding strategies on suspended fibre structures. Glioblastoma cell aggregates were cultured on suspended fibre devices. Fibres guided the outgrowth of cancer cells from the aggregates, mimicking the topography of white matter tracts that assist migration in vivo. Cells assemble into dense (~200 μm depth) tissue structures with necrotic cores, that can remodel the fibre network yet are guided by the underlying fibre organisation. This novel method of patterning suspended microfibres from solution offers several avenues of inquiry to mimic ECM topography and complex material functionality.