Neuron cell culture stands at the forefront of neuroscience innovation, offering unparalleled insights into neuronal development, pathology, and regeneration. This review critically examines advances and persistent obstacles in culturing neurons, with a focus on axon and dendrite growth—a pivotal yet underexplored frontier for regenerative medicine. We synthesize breakthroughs in extracellular matrix (ECM) engineering, 3D biomimetic microenvironments, and molecular interventions while highlighting intrinsic challenges such as limited neuronal longevity, tumorigenicity risks in stem cell approaches, and reproducibility gaps. Introducing a biomimetic engineering framework, we liken neuronal regeneration to a multidimensional optimization problem, where balancing mechanical, biochemical, and epigenetic variables dictates functional outcomes. Key findings include: 1. 3D hydrogels mimicking brain ECM enhance neurite outgrowth by 40–60% compared to 2D systems. 2. Secretory pathway disparities between axons and dendrites reveal evolutionarily conserved growth mechanisms. 3. Tumorigenicity remains a critical barrier, with CRISPR-Lin28-edited iPSCs reducing teratoma formation by 65% in preclinical models. We advocate for standardized, scalable protocols and CRISPR-epigenetic tools to silence inhibitory pathways (e.g., Nogo-A). By bridging in vitro models with clinical translation, this work charts a roadmap for overcoming regenerative bottlenecks in neurodegenerative diseases and CNS injuries.