Human induced pluripotent cells (hiPSCs) can differentiate into various types of brain organoids, which are valuable for applications in tissue engineering and injury repair. Nevertheless, the clinical use of hiPSC-derived cells poses significant challenges due to inconsistency in lineage-specific differentiation. The secreted extracellular vesicles (EVs), in particular the small size subset exosomes (30-200 nm), of hiPSC-derived organoids, have emerged as novel therapeutics in regenerative medicine. In addition to the dimensions and geometry, the viscoelasticity of the hydrogels suitable for organoid engineering is important for studying EV release and its effects in drug delivery. Herein, this study isolated the EVs derived from human spinal cord organoids (hSCOs) after long-term culture (more than 100 days) in bioreactors. In comparison to the static culture, the vertical wheel bioreactor significantly increased the yield of EVs generated from hSCOs, which showed the presence of exosomal markers. The EVs were added to the hSCO differentiation culture at the stage of ventral patterning, and the results reveal that the EVs can regulate hSCO patterning and regional marker expression. Furthermore, hyaluronic acid (HA) hydrogels, which have good water retention and viscoelasticity, can be used as a carrier for sustained-release of EVs. In this study, methylacrylated HA (HAMA) hydrogels were fabricated by different length of crosslinkers (thiolated PEG with linear-2k, 4-arm-4k, and linear-3.4k length), leading to different EV release rates due to different viscoelastic properties (e.g., 97-162 s of stress relaxation). Subsequently, a series of pH-responsive hydrogels (Fe3+-coordinated HAMA@HA-Cat, where HA-Cat refers to dopamine-modified HA) were employed as EV carriers to facilitate hSCO differentiation. As the culture progressed, the pH values of these pH-sensitive hydrogels decreased from 10 to 7 due to CO2 exposure and periodic media changes. This shift in pH altered the viscoelasticity of the hydrogels, thereby modulating EV release kinetics. In addition, EV-loaded HAMA and HAMA@HA-Cat hydrogels were used to regulate hSCO patterning under varying EV concentrations, leading to differential expression of key patterning markers such as DBX1 and ISL1. Finally, the penetration test showed that EVs can pass through the mimicked blood-spinal cord barrier, transporting to the basolateral side. This study also investigated the influence of the EV release in the HA hydrogels for treating an in vitro spinal cord injury model. Taken together, the organoid-secreted EVs in HAMA hydrogels can be released and cross the barrier at the controlled rate and have potential to regulate spinal cord organoid patterning and tissue repair. This study should advance our knowledge of regulating intercellular communication and developing EV-based therapies for treating neurological disorders such as spinal cord injury.
