Air-liquid interface-centered oxygen engineering in human brain organoids: intact, sliced, and microfluidic...

Brain organoids have become essential in vitro models for investigating human brain development, function, and disease, including both unguided cerebral organoids and guided region-specific neural models. However, their utility is fundamentally constrained by oxygen and nutrient diffusion limits inherent to closed three-dimensional architectures. As organoids increase in size and complexity, restricted oxygen delivery induces metabolic stress, disrupts progenitor dynamics, impairs neuronal maturation, and promotes the formation of hypoxic or necrotic cores, thereby limiting developmental fidelity and long-term experimental stability. This review synthesizes current insights into hypoxia and metabolic stress in brain organoid systems, with an emphasis on the physical determinants of gas exchange and their biological consequences. We critically evaluate engineering strategies developed to overcome diffusion-related constraints, focusing on air-liquid interface (ALI) culture and organoid slicing approaches and emerging ALI-microfluidic platforms that integrate controlled perfusion and geometric confinement to actively regulate mass transport. ALI culture improves surface oxygenation while preserving intact tissue architecture, supporting extended viability and functional maturation in intact organoids and assembloids. Sliced organoid platforms directly expose internal tissue compartments, substantially reducing diffusion distances and enabling uniform metabolic conditions that facilitate advanced neuronal differentiation, circuit-level maturation, and high-resolution functional interrogation. Complementing these diffusion-based strategies, ALI-microfluidic systems further enhance metabolic stability and enable dynamic environmental control, scalable organoid production, and integrated electrophysiological assessment under physiologically regulated conditions. By comparing the advantages and limitations of ALI-based systems, this review highlights how oxygen-engineering strategies reshape tissue organization, maturation trajectories, and experimental accessibility, advancing the physiological relevance of human brain organoid models.