Purpose‐Adaptable Reinforced 3D Hyaluronic‐Acid Based Platform to Study Pathomechanisms of the Central Nervous System

Three-dimensional (3D) models to study human disease mechanisms have demonstrated that the third dimension is an essential component for neuronal maturation and function. However, 3D neuronal cell culture is challenging due to their ultra-soft nature and specific extracellular matrix (ECM) organization. This study presents a microfiber reinforcement approach, combining primary mouse spinal cord neurons (SCNs) in a purpose-adaptable hyaluronic-acid-based matrix with melt electrowritten (MEW) frames to study disease mechanisms. The importance of laminins (LNs) is evaluated, which are vital for neuronal adhesion and maturation. Astrocytes (ACs) are mandatory in brain development and function by secretion of signal molecules, maintaining ion homeostasis, clearing of neurotransmitters, and by actively modulating neuronal activity. Three combinations are compared (i) isolated primary SCN, (ii) SCN with ACs (SCN-AC) and (iii) SCN-AC and LNs (SCN-AC-LN). Multimodal analysis by comparing protein expression, dendrite length, complex mechanical properties, and network functionality via Ca²⁺-imaging allows validation of structural and functional neuronal network development. The suitability of the 3D model to study pathomechanisms is demonstrated for Stiff Person Syndrome (SPS). For the first time, functional impairment of spinal cord neurons by patient-derived autoantibodies characteristic for SPS is recapitulated in a 3D spinal cord model platform adaptable to other disease types.