Free-Standing and Aligned Hydrogel Nanofibers Sheet for 3D Cell Culture

Abstract

Synthetic scaffolds aim to emulate extracellular matrix (ECM), which is present in all tissues, and it provides structural support and maintains cellular microenvironment. Micro-nano fibrillar hydrogel has been considered a versatile material for designing synthetic scaffolds because of its biocompatibility, low mechanical moduli, controllable water retention, and microfeatures sizes. An additional feature of ECM is anisotropy because anisotropic structures play a significant role in biological systems. However, conventional fabrication methods such as electrospinning develop micro-nanofibers with randomly oriented fiber mesh. Furthermore, two-dimensional anisotropic scaffolds developed by shear-force assisted alignment onto substrates differ from biological systems, which are anisotropically fashioned in three dimensions (3D). Therefore, anisotropic 3D and substrate-free scaffolds are needed to investigate further particular cellular functions and potential uses for in vivo implantation experiments. In this work, hydrogel nanofibers (HNFs) were aligned by bar coating and fixed on substrates by layer-by-layer assembly with the assistance of a gelatin sacrificial interlayer. The gelatin sacrificial interlayer supports obtaining a programmable releasement of free-standing films. Moreover, HNFs deposited onto gelatin showed well-maintained anisotropy before and after detachment from the supporting substrates. Consequently, freeze/thaw and freeze/soak processes were employed to preserve the directionality and mechanical properties of free-standing HNFs sheets. Finally, aligned HNFs were framed by a PET fixture for efficient handling, showing minimal cytotoxicity confirmed by live and dead assay. Primary cultured cardiomyocytes and neurons on free-standing HNFs sheets showed good viability and 3D morphology with directional alignment along underlying fibers.