Organ-on-Chips (OoCs) are microfluidic cell culture devices which offer a controlled in vivo mimicking in vitro platform in order to study the dynamics, functionality and pathophysiological behavior of a wide range of biological entities (from single cells to tissues and especially engineered (sub-)organs). Similarly, the applications vary from biological basic research and personalized medicine to toxicological tests and drug development, where OoCs may also reduce animal studies. To gain full control of the cell culture conditions as well as more characterization and stimulation options, the OoC’s can be integrated with recording and/or stimulation electrodes and chemical or physical sensors; these are connected to required environment controllers such as heating plates. Complex microfluidic tunnels and compartments have an important and versatile role in providing not only nutrients for the cell culture, but also drugs, other chemical stimulants, gassing, as well as in restricting and/or guiding the cell growth. Factors such as emerging 3D cell cultures, multi-organ systems (i.e. Body-on-Chips), and vascularization (etc.) set new requirements for the OoC technology. This JoVE Methods Collection aims to answer these challenges and awaits submissions dealing especially with integrating electrodes, sensors, and various stimulation methods in 3D (and also 2D) cell cultures as well as: (1) novel microfluidic device designs; (2) concepts and fabrication methods; and (3) other technical demonstrations related to Organ-on-Chips.