Associate Professor Indiana University Bloomington Bloomington, Indiana, United States
Introduction: : Human brain organoids, 3D brain-like tissues derived from human stem cells, can replicate key features of brain development, offering great potential for studying neurodevelopmental, neurodegenerative, and neuropsychiatric diseases, as well as for drug discovery and regenerative medicine. Although cerebral organoids and other region-specific models have advanced the field, they still fall short of fully mimicking in vivo brain physiology. A major limitation is the lack of vascularization, leading to poor oxygen and nutrient diffusion, resulting in stressed, hypoxic, and necrotic cores that impair development and maturation, causing variability and reduced reproducibility.
To overcome diffusion limitations, strategies such as in vivo transplantation into rodent hosts have been explored, supporting organoid survival and integration with host brains. Alternatively, organotypic slice cultures at the air-liquid interface improve diffusion and preserve organoid morphology. Mechanical spinning systems have further enabled the long-term culture of sliced neocortical organoids, promoting neurogenesis and cortical plate formation. Engineering approaches like organ-on-a-chip and vascularized scaffolds have also been developed to enhance diffusion using microfabricated devices. Despite these advances, creating simple, robust methods remains challenging for broad laboratory applications.
Inspired by the brain’s vascular network, we present vascular network-inspired diffuse (VID) scaffolds to enhance neural organoid development. Fabricated through cost-effective 3D printing of biocompatible plastics, VID scaffolds emulate natural diffusion processes, significantly reducing apoptosis, relieving cellular stress, sustaining neurogenesis, and promoting functional maturation and drug responsiveness. VID scaffolds can be seamlessly integrated into existing protocols, providing a powerful new tool for engineering more functional and reproducible brain organoids.
Materials and
Methods: : The human ESCs (WA09) were obtained from the WiCell Institute, following the guidelines of both the WiCell Institute and the Biosafety Committee of Indiana University. VID scaffolds were designed using AutoCAD and fabricated with a stereolithographic 3D printer. Scaffolds were coated with poly-D-lysine and laminin before use. Conventional midbrain organoids and engineered midbrain organoids were generated from WA09 cells. Engineered midbrain organoids incorporated VID scaffolds from day 3 of culture. Organoids were cryosectioned, immunostained, and imaged using confocal and inverted microscopes. Fontana–Masson staining and dopamine ELISA assays were performed to assess pigmentation and dopamine production. Perfusion assays and hypoxia staining were used to evaluate scaffold function and oxygen diffusion. Single-cell RNA sequencing was conducted following papain-based dissociation, and data were analyzed using Seurat. For functional studies, organoids were plated onto MEA plates to record spontaneous neural activity, and functional connectivity was assessed using STTC analysis. Fentanyl treatments were used to evaluate drug responsiveness. Flow simulations were performed in COMSOL to model oxygen perfusion within scaffolds.
Results, Conclusions, and Discussions:: We developed and fabricated VID scaffolds that, combined with orbital shaking-induced flows, recreate physiological diffusion for organoid culture (Figure 1). Using these scaffolds, we successfully generated engineered midbrain organoids —flattened, functional human midbrain organoids with diffusible tube networks—from stem cells (Figure 1). The engineered midbrain organoids showed enhanced cell survival, reduced stress, sustained neurogenesis, stronger region-specific differentiation, and greater neural activity compared to conventional midbrain organoids (Figure 2). Notably, engineered midbrain organoids also displayed improved neural responses to fentanyl, unlike conventional midbrain organoids (Figure 3). These scaffolds offer a promising platform for engineering and phenotyping functional organoids under physiological perfusion-like conditions. Our VID scaffolds provide several advantages: they fully replicate vascular diffusion, supporting oxygen, nutrient, and signaling delivery throughout development and maturation. The biocompatible, 3D-printed plastic scaffolds enable prolonged organoid survival and function. Their flattened design maintains most cells within diffusion limits and allows compatibility with MEA chips and confocal imaging. Scaffold dimensions are customizable to fit standard 24-, 96-, or 384-well plates, supporting easy integration with existing protocols. Furthermore, simple and cost-effective 3D printing allows scalable production. Lastly, engineered midbrain organoids generated on these scaffolds reproduce classical pharmacological responses, underscoring their potential for drug screening and standardized organoid generation.
Acknowledgements and/or References (Optional):: This study was supported by the National Institute of Health Awards (DP2AI160242 and U01DA056242). We also acknowledge the Indiana University Imaging Center (NIH1S10OD024988-01). For more details, please see our publication on Cell Stem Cell, 2025 (DOI: 10.1016/j.stem.2025.02.010)
