Mastering Organoid Growth: A Complete Guide to Overcoming Methodological Challenges

Organoid technology has become among the most popular technologies in recent years, due to their three-dimensional and physiologically enriching models that closely mimic the structure and function of human organs. Herein, this review details the in-depth methodology, updated to date, for the efficient cultivation of organoids. Emphasizing liver organoids, both hepatocyte and cholangiocyte derived, and other abdominal organ systems, such as gut, kidney, and pancreas, we explore the technological challenges researchers are facing nowadays, including how to optimize nutrient delivery, maintain cellular diversity, achieve scalability in the organoid culture system, and high-throughput applications. Addressing those biological and technological complexities, this review aimed at equipping new researchers with practical insights and standardized protocols that will help improve reproducibility and success rates in organoid culture and expand their applications. Furthermore, we discuss current limitations and barriers to clinical translation, highlight key knowledge gaps, and outline emerging innovations, including bioengineering, microfluidic systems, and genetic manipulation, expected to further enhance disease modeling, personalized medicine, and regenerative therapies. Finally, we provide perspective on next-generation technologies that expedite organoid-based discovery and development.