This book provides an overview of established 3D cell culture assays from leaders in the field. Their contributions cover a wide spectrum of techniques and approaches for 3D cell culture, from organoid cultures through organotypic models to microfluidic approaches and emerging 3D bioprinting techniques, which are used in developmental, stem cell, cancer, and pharmacological studies, among many others. Written for the highly successful Methods in Molecular Biology series, 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.  Comprehensive and cutting-edge, 3D Cell Culture: Methods and Protocols aims to inspire researchers to develop novel 3D cell culture techniques according to their specific scientific needs and interests, leading to a new generation of physiologically relevant and realistic 3D cell cultures. Chapter 15 of this book is available open access under a CC BY 4.0 license.