Synergistic Effects of Red Light and Direct Current Electric Fields on Neurite Growth in 3D Neural Cell Cultures

Both red-light illumination and direct-current electric fields (dcEFs) have independently been shown to promote neurite outgrowth in two-dimensional (2D) neural cell cultures. However, their combined effects in a three-dimensional (3D) culture environment remain unexplored. In this study, we examined the synergistic effects of red light and dcEFs on neurite extension in human neuroblastoma (SH-SY5Y) and mouse neuroblastoma (N2a) cells embedded within a 3D collagen gel matrix. Our results demonstrated that optimized dcEF stimulation and red-light exposure significantly enhanced neurite elongation and directional alignment. RNA sequencing further identified Neuropeptide Y (NPY) and its receptors as key mediators of these effects. Moreover, electrophysiological assessments revealed that neurons subjected to the combined stimulation exhibited enhanced functional maturation. These findings provide compelling evidence supporting the potential applications of red light and dcEFs in neural regeneration and repair. Overall design: This study investigates the combined effects of direct current electric field (175 mV/mm, 3 hours) and red light (620 nm, 2 mW/cm², 45 hours) stimulation on neurite growth in 3D SH-SY5Y neural cell cultures. Cells were embedded in 3D collagen scaffolds at a concentration of 1 mg/mL, with a seeding density of 1×106 cells/mL. Four experimental groups were included: (1) Control (no treatment), (2) dcEF only, (3) Red light only, and (4) Combined dcEF and red light. Total RNA was extracted after treatment. Two independent batches of RNA-seq libraries were generated using the same experimental design and were jointly analyzed to assess reproducibility and treatment effects. Each group included biological triplicates. Sequencing was performed using the Illumina NovaSeq X Plus platform with paired-end reads.