An in vitro model using spheroids-laden nanofibrous structures for attaining high degree of myoblast alignment and differentiation

A spheroid is an aggregation of single cells with structural and functional characteristics similar to those of 3D native tissues, and it has been utilized as one of the typical in vitro three-dimensional (3D) cell models. Scaffold-free spheroids provide outstanding reflection of tissue complexity in a 3D in vivo-like environment, but they can neither fabricate realistic macroscale 3D complex structures without avoiding necrosis nor receive direct external stimuli (i.e., stimuli from mechanical or topographical cues). Here, we propose a spheroid-laden electrospinning process to obtain in vitro model achieved using the synergistic effect of the unique bioactive components provided by the spheroids and stimulating effects provided by the aligned nanofibers. Methods: To show the functional activity of the spheroid-laden structures, we used myoblast-spheroids to obtain skeletal muscle, comprising highly aligned myotubes, utilizing an uniaxially arranged topographical cue. The spheroid-electrospinning was used to align spheroids directly by embedding them in aligned alginate nanofibers, which were controlled with various materials and processing parameters. Results: The spheroids laden in the alginate nanofibers showed high cell viability (>90%) and was compared with that of a cell-laden alginate nanofiber that was electrospun with single cells. Consequently, the spheroids laden in the aligned nanofibers showed a significantly higher degree of myotube formation and maturation. Conclusion: Results suggested that the in vitro model using electrospun spheroids could potentially be employed to understand myogenic responses for various in vitro drug tests.