Synergistic Enhancement of Peripheral Nerve Regeneration via Ibudilast-Primed Three-Dimensional Spheroid Culture of Human Adipose-Derived Stem Cells

TL;DR

This study shows that combining 3D cell culture with the drug ibudilast boosts the regenerative power of fat-derived stem cells for nerve repair.

Contribution

A safe, synergistic strategy using 3D spheroids and ibudilast to enhance stem cell secretions for nerve regeneration.

Findings

Ibudilast-primed 3D spheroids increased NGF, VEGF, and IGF-1 secretion 6- to 14-fold compared to 3D alone.

Conditioned media from these spheroids accelerated human Schwann cell migration and upregulated myelination genes.

Trophic effects from the primed spheroids lasted up to 72 hours.

Abstract

Background: Peripheral nerve regeneration relies on Schwann cell activation and neurotrophic support. Although adipose-derived stem cells (ADSCs) show therapeutic potential through paracrine mechanisms, their clinical application is often limited by donor-dependent heterogeneity in therapeutic efficacy. Accordingly, strategies to standardize and potentiate their secretory function are essential. This study investigated a safety-optimized strategy to achieve this by combining three-dimensional (3D) spheroid culture with ibudilast, a clinically approved phosphodiesterase inhibitor. Methods: Human ADSCs were cultured in 2D or 3D conditions with varying ibudilast concentrations. Safety was confirmed via CCK-8 assays, and trophic factor secretion was quantified by RT-qPCR and ELISA. To rigorously validate functional outcomes, conditioned media were applied to a dual-model system comprising immortalized rat (RSC96) and primary human Schwann cells (HSwCs), assessing migration and the expression of regeneration-associated genes. Results: Ibudilast demonstrated no cytotoxicity. While 3D culture alone enhanced secretion compared to 2D controls, the addition of ibudilast provided a synergistic boost, resulting in a 6- to 14-fold increase in NGF, VEGF, and IGF-1 levels compared to 3D spheroids alone. Notably, conditioned media from these primed spheroids significantly accelerated HSwCs migration and induced robust upregulation of myelination-related genes (specifically PMP22 and EGR2), with trophic effects sustained for up to 72 h. Conclusions: Ibudilast-primed 3D spheroids synergistically amplify the neuroregenerative secretome of ADSCs. By utilizing a repurposed, safe small molecule to overcome functional variability and maximize potency without genetic manipulation, this strategy represents a highly translatable candidate for peripheral nerve repair.

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