Enhanced Neural Differentiation Using Simultaneous Application of 3D Scaffold Culture, Fluid Flow, and Electrical...

Neural differentiation is studied using a simultaneous application of 3D scaffold culture and hydrodynamic and electrical stimuli in purpose-designed recirculation bioreactors operated with continuous fluid flow. Pheochromocytoma (PC12) cells are seeded into nonwoven microfibrous viscose-rayon scaffolds functionalized with poly-l-lysine and laminin. Compared with the results from static control cultures with and without electrical stimulation and bioreactor cultures with the fluid flow without electrical stimulation, expression levels of the differentiation markers β3-tubulin, shootin1, and ephrin type-A receptor 2 are greatest when cells are cultured in bioreactors with fluid flow combined with in-situ electrical stimulus. Immunocytochemical assessment of neurite development and morphology within the scaffolds confirm the beneficial effects of exposing the cells to concurrent hydrodynamic and electrical treatments. Under the conditions tested, electrical stimulation by itself produces more pronounced levels of cell differentiation than fluid flow alone; however, significant additional improvements in differentiation are achieved by combining these treatments. Fluid flow and electrical stimuli exert independent and noninteractive effects on cellular differentiation, suggesting that interference between the mechanisms of differentiation enhancement by these two treatments is minimal during their simultaneous application. This work demonstrates the beneficial effects of combining several different potent physical environmental stimuli in cell culture systems to promote neurogenesis.