Since the publication of the previous edition, the use of 3D cell and organoid cultures has become more widespread across laboratories. This second edition volume expands on the previous edition with discussions about the latest organoid models developed for many more organs; new hydrogels and devices for 3D culture; and the organoid systems that have been improved by incorporating more components of tissue microenvironments in the in vitro culture. The chapters in this book are organized into five parts and cover topics such as biofabrication, organoids, microfluidic systems, bioprinting, and image analysis. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Thorough and cutting-edge, 3D Cell Culture: Methods and Protocols, Second Edition is a valuable resource that will stimulate new ideas, foster interdisciplinary collaborations, and contribute to the improvement of human health and well-being.