Title : Evaluating an electrically stimulated RN22– NG108-15 co-culture platform for peripheral nerve tissue engineering
Abstract:
Introduction: Peripheral nerve regeneration remains challenged by the limited effectiveness of current clinical repair strategies, including autologous nerve grafting. The development of physiologically relevant in vitro models that reproduce neuron–Schwann cell interactions while incorporating bioelectrical cues is essential for advancing peripheral nerve tissue engineering. Although electrical stimulation is recognized as a regulator of neuronal behavior, its application within multicellular peripheral nerve models remains insufficiently explored.
Objectives: To develop and evaluate an electrically stimulated RN22–NG108-15 co-culture as an in vitro platform for peripheral nerve tissue engineering..
Methods: RN22 Schwann-derived cells and NG108-15 neuron-like cells were individually characterized for viability and proliferation prior to establishing a co-culture using an optimized hybrid culture medium. Electrical stimulation was applied through integrated electrodes under standardized stimulation conditions. Cell viability was evaluated using the WST-1 assay, while optical and immunofluorescence microscopy were employed to assess cell morphology, neurite development, phenotype preservation, and neuron–Schwann cell interactions.
Results: The optimized co-culture maintained high viability and stable growth, demonstrating compatibility between both cell populations throughout the experimental period. Immunofluorescence analysis confirmed preservation of both Schwann and neuronal phenotypes under co-culture conditions. Electrical stimulation induced a significant improvement in cellular behavior, evidenced by enhanced neuronal differentiation, increased neurite extension and neurite network formation, and improved neuron–Schwann cell interactions compared with unstimulated cultures. Furthermore, a positive relationship was observed between electrical stimulation and physiological cellular behavior, as demonstrated by improvements in cell viability and neurite-related parameters, supporting the beneficial role of bioelectrical stimulation in promoting a regenerative cellular microenvironment.
Conclusions: The proposed electrically stimulated RN22–NG108-15 co-culture constitutes a reproducible in vitro platform that reproduces key cellular features of the peripheral nerve microenvironment. By integrating multicellular neuron–Schwann cell interactions with controlled electrical stimulation, this model provides a versatile platform for investigating peripheral nerve regeneration mechanisms, evaluating biomaterials and regenerative therapies, and supporting the development of tissue engineering strategies while contributing to the reduction of animal experimentation.
