Organoids: generation strategies, applications, and future challenges

Organoids are microscopic 3D structures that resemble real organs, assembled in vitro from stem cells. Since the Dutch team of Hans Clevers successfully cultured intestinal stem cells to generate intestinal structures in 2009, organoid technology has developed rapidly, and culture protocols covering various organs such as the brain, liver, intestine, kidney, and bone have been established. These micro-organ models retain the cellular heterogeneity, tissue-specific structure, and genetic background of the original tissue. Compared to traditional two-dimensional culture, they provide a more physiologically relevant research platform. This article reviews organoid generation strategies, mainly dividing them into scaffold-free and scaffolded methods, and details specific generation protocols for brain, liver, intestine, kidney, and bone organoids. Furthermore, this article emphasizes innovations in bioengineering, such as organ-on-a-chip systems and 3D bioprinting technology. These technologies can enhance the maturity, vascularization, and reproducibility of organoids. The article also extensively discusses the biomedical applications of organoids, including in vitro disease models, infectious diseases, cancer, drug screening and toxicity testing, tissue engineering, and age-related diseases. Despite the significant potential of organoids, some challenges remain. Issues such as standardization, limited vascularization, ethical considerations, and scalability for industrial and clinical translation remain. Looking ahead, interdisciplinary efforts integrating stem cell biology, bioengineering, and computational methods promise to drive the development of organoid technology. This will make it a more robust and physiologically consistent model. Organoids hold the potential to become a cornerstone tool in biomedical research, bridging the gap between in vitro research and the clinical application of personalized medicine, drug development, and regenerative therapies.