Integrated Microfluidic Hydrogel-Enhanced Microwell Array for Uniform Self-Assembly, Long-Term Culture, and Neural Differentiation of Mesenchymal Stem Cell Spheroids

3D culture of mesenchymal stem cells (MSCs) more effectively supports spheroid formation and sustained growth factor secretion, creating a microenvironment conducive to neural differentiation compared to traditional monolayer culture. However, challenges in long-term culture, including heterogeneity, central necrosis, and scalability, hinder its application in neural regeneration therapies. This study presents an automated microfluidic hydrogel-enhanced spheroid formation and maintenance array (M-HESMA) chip, combining a basement membrane-like hydrogel, microwell array, and dynamic culture system. The hydrogel, comprising a dual-network of Pluronic F-127-heparin (PF127-Hep) and crosslinked hyaluronic acid (XL-HA) with growth factors, supports spheroid formation and growth. The M-HESMA chip is validated using stem cells from human exfoliated deciduous teeth (SHEDs), forming uniform spheroids with minimal necrosis. The derived neurospheroids (MSNSs) exhibit enhanced neural differentiation and paracrine potential. Dissociated MSNSs (MSNS diss ) are applied to 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) mice, improving motor function and reducing pathological symptoms compared to monolayer-cultured SHEDs treatment. MSNS diss -treated mice show increased brain-derived neurotrophic factor (BDNF) levels, phosphoinositide 3-kinase/protein kinase b (PI3K/Akt) pathway activation, and decreased neuronal apoptosis. The M-HESMA chip provides a scalable, efficient platform for spheroid generation, offering a promising approach for MSCs-based therapies targeting neurodegenerative diseases.